Amplification of assay reporters by nucleic acid replication

ABSTRACT

PCT No. PCT/US93/01281 Sec. 371 Date Feb. 13, 1995 Sec. 102(e) Date Feb. 13, 1995 PCT Filed Feb. 4, 1993 PCT Pub. No. WO93/15229 PCT Pub. Date Aug. 5, 1993A method for the amplified detection of an analyte, wherein amplification is achieved by replication of a target nucleic acid sequence which has been immobilized in response to analyte.

This application is a 371 of PCT/US 93/01281 filed on Feb. 4, 1993.

FIELD OF THE INVENTION

This invention relates to a method for the amplified detection of ananalyte in fluid, wherein amplification is achieved by replicating atarget nucleic acid sequence which has been immobilized in response toanalyte.

BACKGROUND OF THE INVENTION

The introduction of immunoassays in the 1960s and 1970s greatlyincreased the number of analytes amenable to precise and accuratemeasurement. Radio immunoassays (RIAs) and immunoradiometric (IRMA)assays utilize radioisotopic labeling of either an antibody or acompeting antigen to measure an analyte. Detection systems based onenzymes or fluorescent labels were then developed as an alternative toisotopic detection systems. D. L. Bates, Trends in Biotechnology, 5(7),204 (1987), describes one such method based upon enzyme amplification.In this method a secondary enzyme system is coupled to a primary enzymelabel, for example, the primary enzyme can be linked catalytically to anadditional system such as a substrate cycle or an enzyme cascade. Enzymeamplification results from the coupling of catalytic processes, eitherby direct modification or by interaction with the product of thecontrolling enzyme.

U.S. Pat. No. 4,668,621 describes utilization of an enzyme-linkedcoagulation assay (ELCA) in an amplified immunoassay using a clottingcascade to enhance sensitivity. The process involves clot formation dueto thrombin activated fibrin formation from soluble fibrinogen andlabeled solubilized fibrinogen. Amplification of the amount ofreportable ligand attached to solid phase is obtained only by combininguse of clotting factor conjugates with subsequent coagulation cascadereactions.

Substrate/cofactor cycling is another variation of enzyme-mediatedamplification, and is based on the cycling of a cofactor or substratewhich is generated by a primary enzyme label. The product of the primaryenzyme is a catalytic activator of an amplifier cycle which responds inproportion to the concentration of substrate and hence the concentrationof the enzyme label. An example of this type of substrate cycling systemis described in U.S. Pat. No. 4,745,054.

Vary et al., Clin Chem., 32, 1696 (1986) describes an enzymeamplification method suited to nucleic acid detection. This method is astrand displacement assay which uses the unique ability of apolynucleotide to act as a substrate label which can be released by aphosphorylase.

Bobrow et al., J. of Immunol. Methods, 125, 279 (1989) discloses amethod to improve detection or quantitation of an analyte by catalyzedreporter deposition. Amplification of the detector signal is achieved byactivating a conjugate consisting of a detectably labeled substratespecific for the enzyme system, wherein said conjugate then reacts withthe analyte-dependent enzyme activation system to form an activatedconjugate which deposits wherever receptor for the conjugate isimmobilized.

Nucleotide hybridization assays have been developed as a means fordetection of specific nucleic acid sequences. U.S. Pat. No. 4,882,269discloses an amplified nucleic acid hybridization assay in which atarget nucleic acid is contacted with a complementary primary probehaving a polymeric tail. A plurality of second signal-generating probescapable of binding to the polymeric tail are added to achieve amplifieddetection of the target nucleic acid. Variations of this methodology aredisclosed in PCT Application WO 89/03891 and European Patent Application204510, which describe hybridization assays in which amplifier ormultimer oligonucleotides are hybridized to a single-stranded nucleicacid unit which has been bound to the targeted nucleic acid segment.Signal amplification is accomplished by hybridizing signal-emittingnucleic acid bases to these amplifier and multimer strands. In all ofthese disclosures amplification is achieved by mechanisms whichimmobilize additional sites for attachment of signal-emitting probes.

In contrast, the present invention utilizes a fundamentally differentconcept in achieving signal amplification. In response to analyte, atarget nucleic acid sequence is immobilized and replicated using nucleicacid replication techniques. Signal enhancement is achieved bygenerating and detecting replicates of the target sequence.

U.S. Pat. No. 4,994,368 discloses a nucleic acid hybridization assaywhich accomplishes detection of polynucleotide analytes by producingreplicated copies of a primary polynucleotide sequence. The targetsequence of interest is first restricted to provide a free 3' OH end,and then is hybridized to a complementary binding sequence located atthe 3' end of two or more template sequences in a single-strandedpattern polynucleotide. Chain extension is performed on the targetsequence, and this extension product is then cleaved into fragmentswhich are subsequently hybridized with single-stranded patternnucleotide. The polymerization, cleavage, rehybridization,polymerization cycle is repeated until a detectable number of copieshave been obtained. In a similar vein, PCT application WO 90/0345describes a nucleic acid detection assay wherein the reporter moleculeis an adduct comprising 1) an oligonucleotide probe sequence which iscomplementary to the targeted site; 2) a primer sequence capable ofinitiating primer extension; and 3) a sequence segment which iscomplementary to the primer sequence. As initially added to the testnucleic acid sample, the adduct assumes a hairpin structure whichrenders the primer inactive. Upon hybridization of the adduct to atarget sequence in the sample, however, the adduct becomes activated andits primer sequence becomes available for initiating a primer extensionproduct. The art methods differ from that of Applicants' in that the artuses significantly different and more cumbersome approaches to producingmultiple copies of a detectable nucleic acid. Also, these methods arelimited to the detection of nucleotide sequences, while Applicants'method is applicable to a wide range of analytes.

The use of RNA as a reporter for immunolocical assays has been describedin the literature. WO 87/06270 teaches the use of an RNA capable ofbeing autocatalytically replicated by an RNA-dependent RNA polymerase asa reporter for assaying biopolymers by immunoassay or by nucleic acidprobe hybridization.

Similarly WO 91/17442 describes various protein/nucleic acid hybridprobes which can be used to amplify the detectable signal inimmunoassays. Signal is amplified by a method comprising firstimmobilizing an antigenic analyte on a solid substrate, binding to theanalyte a protein/nucleic acid hybrid probe comprising a double-strandedRNA T7 polymerase promoter operably connected to either asingle-stranded or double-stranded nucleic acid template, removing anyunbound probe, transcribing multiple copies of RNA oligomers anddetecting and quantifying the transcripts. Template replication is onthe order of 10¹ to 10⁴ copies per template.

The above methods are useful for enhancing the level of detection ofanalytes by immunoassay, however, both methods suffer from significantrestrictions. For example, both methods rely on the use of RNA-dependentpolymerases for nucleic acid replication which gives inherently lessamplification than other nucleic acid amplification methods such asPolymerase Chain Reaction (PCR) or Ligase Chain Reaction (LCR), and doesnot result in a molecularly-defined product. It is well known in the artthat PCR, for example, will give amplification on the order of 10⁶ to10¹⁴ copies per target of discreet length. Furthermore, the abovemethods are not easily adapted to the detection of more than one analytein a sample.

Sano et al. Science, 258, 120, (1992) describes an antigen detectionsystem, termed Immuno-PCR, in which a specific DNA molecule is used as areporter. A streptavidin-protein A chimera was used to attach abiotinylated DNA to antigen-monoclonal antibody complex that had beenimmobilized on microtiter plate wells. A segment of the complexed DNAwas amplified by Polymerase Chain Reaction (PCR) and the PCR productswere analyzed by gel electrophoresis. This method is limited by the needfor multiple reagent additions and extensive washing requirements.

SUMMARY OF THE INVENTION

Applicants disclose a sensitive method for detecting an analyte byamplifying the detectable response of an analyte-dependent reportersystem. The amplification is achieved using nucleic acid replication ofa target nucleic acid sequence after said target sequence has beenimmobilized in response to the presence of an analyte.

The invention is an amplified detection method for the detection andquantitation of an analyte in a fluid sample. The method comprises firstimmobilizing an analyte to form what Applicants have termed an"analyte-dependent reporter system" (ADRS). The ADRS will be comprisedof a target nucleic acid sequence which has been immobilized in responseto the presence of analyte in the sample. Next, the immobilized targetnucleic acid sequence of the ADRS is contacted with a nucleic acidreplication composition under conditions wherein the target sequence maybe replicated, and replication of the target is carried out. Andfinally, the replicated target nucleic acid sequences are detected,whereby the presence of analyte may be determined.

Applicants' amplified detection method may be specifically designed tobe practiced in a number of different ways. For example, Applicants havepresented four possible variations of the method (see FIGS. 1, 2, 3, and6).

In a preferred embodiment of Applicants' method, reference nucleic acidsequences which are different from the target sequences would beincluded with the ADRS at one or more steps and replicated concurrentlywith the target sequences under the assay conditions of that method.These reference sequences will be designed to generate sequences whichare detectably distinct from the replicated target sequences, and willtherefore serve as measures of internal control for each particularassay methodology.

In another preferred embodiment, which is illustrated in FIGS. 4 and 5,the method can be used to detect several analytes within one sample byvarying the length of the "variable segment" of the target nucleic acidused in the reporter conjugate for each analyte. In this way, the sameprimers may be used for all replications within one sample, and sizeseparation of the replicated nucleic acid targets will enable convenientdetection of multiple analytes in one sample.

In still another embodiment, which is illustrated in FIG. 6, aconvenient variation of the method is disclosed wherein a ligandreporter conjugate is used to compete with sample analytes for bindingsites on the capture reagent. Target segments on the unbound ligandreporter conjugates may then be replicated, indicating the presence ofanalytes in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the "Direct Target Deposition Method" of amplifyingthe response of an analyte-dependent reporter system.

FIG. 2 illustrates the "Catalyzed Target Deposition Method" ofamplifying the response of an analyte-dependent reporter system.

FIG. 3 illustrates the "Catalyzed Indirect Target Deposition Method" ofamplifying an analyte-dependent reporter system.

FIG. 4 illustrates the use of the "Direct Target Deposition Method" todetect more than one analyte per sample.

FIG. 5 illustrates a variable length reporter conjugate designed for usein the "Direct Target Deposition Method" to detect more than one analyteper sample.

FIG. 6 illustrates the "Competitive Binding Method" of amplifying ananalyte-dependent reporter system.

FIG. 7 is a photograph of an electrophoretic agarose gel used toseparate the replicated target sequences in a "Direct Target Deposition"assay of the instant invention.

DETAILED DESCRIPTION OF THE INVENTION

Applicants disclose a sensitive method for detecting an analyte byamplifying the detectable response of an analyte-dependent reportersystem. The amplification is achieved using nucleic acid replication ofa target nucleic acid sequence in response to the presence of ananalyte.

The present invention provides a method for amplifying the response ofan analyte-dependent reporter complex (ADRC). The ADRC is formed inresponse to analyte and contains the bound analyte. The ADRC maydirectly immobilize a target nucleic acid, or may react with otherreagents to ultimately result in target nucleic acid sequences which areimmobilized onto receptors. The resulting product is a nucleic acidreplication system which is capable of forming many copies of a targetnucleic acid sequence when the proper replication reagents are added.The whole immobilized replication system is referred to by Applicants asthe analyte-dependent reporter system (ADRS). Copies of the resultingamplified nucleic acids from the ADRS may be detected, providing a meanswhich is useful for the detection and measurement of analytes in testfluid.

The term "analyte" will refer to a substance to be detected or assayedby the method of the present invention. Typical analytes may include,but are not limited to proteins, peptides, nucleic acid segments,molecules, cells, microorganisms and fragments and products thereof, orany substance for which attachment sites, binding members or receptors(such as antibodies) can be developed.

The term "analyte-dependent reporter system" (ADRS) refers to animmobilized system which is formed in response to the presence of ananalyte. The system will contain an immobilized nucleic acid targetsequence. The analyte is subsequently detected or quantitated bydetection of amplified copies of this target nucleic acid sequence.

The term "analyte-dependent reporter complex" (ADRC) refers to onecomponent of the above system. The ADRC refers to an immobilized analytecapture reagent, an analyte, and a reporter conjugate.

The term "immobilized capture reagent" refers to any substance capableof binding an analyte, such as an antibody, receptor, lectin, nucleicacid or binding protein, which has been immobilized by attachment to anappropriate support. Also, in some instances, the immobilized capturereagent may simply be comprised of a solid support matrix to which ananalyte may bind without the aide of an intermediary substance.

The term "reporter conjugate" refers to either: 1) a conjugatecomprising a target nucleic acid sequence coupled to one member of abinding pair such as an antibody, lectin, receptor or binding protein orother moiety which can bind to analyte (as in FIG. 1); or alternatively2) to a conjugate comprising an enzyme coupled to one member of abinding pair such as an antibody, lectin, receptor or binding protein(as in FIGS. 2 and 3).

The term "target nucleic acid sequence" or "target sequence" or "target"refers to the template nucleic acid within the ADRS which will bereplicated to generate replicated nucleic acid target sequences.

The term "nucleic acid replication composition" refers to a compositioncomprising the ingredients necessary for performing nucleic acidreplication. Applicants contemplate that replication may be accomplishedby any of several schemes known in this art, including but not limitedto the polymerase chain reaction (PCR); or the ligase chain reaction(LCR). If PCR methodology is selected, the replication composition wouldinclude for example, nucleotide triphosphates, two primers withappropriate sequences, DNA or RNA polymerase and proteins. Thesereagents and details describing procedures for their use in amplifyingnucleic acids are provided in U.S. Pat. No. 4,683,202 (1987, Mullis, etal.) and U.S. Pat. No. 4,683,195 (1986, Mullis, et al.), which arehereby incorporated by reference. If LCR methodology is selected, thenthe nucleic acid replication compositions would comprise, for example, athermostable ligase, e.g., T. aquaticus ligase, two sets of adjacentoligonucleotides wherein one member of each set is complementary to eachof the target strands, Tris HCl buffer, KCl, EDTA, NAD, dithiothreitoland salmon sperm DNA. See, for example, Tabor, S. and Richardson, C. C.(1985) Proc. Acad. Sci. USA 82, 1074-1078), which is hereby incorporatedby reference.

The term "replicated target sequence" refers to the copies of the targetnucleic acid sequence produced in the replication process.

The term "nucleic acid replication substrate" refers to a conjugatecomprising a target nucleic acid sequence connected, optionally via aspacer, to a moiety capable of activation by an enzyme.

The term "activated nucleic acid replication intermediate" refers to theproduct obtained from reaction of the nucleic acid replication substratewith the enzyme of the ADRC.

The term "deposited nucleic acid replication product" refers to theproduct resulting from deposition of the activated nucleic acidreplication intermediate onto a receptor.

The term "deposit" or "deposition" means directed binding to animmobilized receptor. Such deposition may result for example, fromformation of a covalent bond, direct binding to a solid matrix, or froma specific binding pair interaction.

The term "receptor" means any site which will bind to an activatedconjugate of the ADRS, either through the formation of a covalent bondor through a specific binding pair interaction. For example, in SchemesII and III (see FIGS. 1 and 2), the receptors for the activatedconjugates of step 2 may be located on the same support whichimmobilizes the ADRC (as shown); or alternatively, the receptors for theactivated conjugates of step 2 may be located on different insolublesupports.

The term "binding substrate" refers to a conjugate comprising a firstmember of a binding pair species, optionally a spacer, and a moietycapable of being activated by an enzyme.

The term "binding pair" includes any of the class of immune-type bindingpairs, such as antigen/antibody or hapten/anti-hapten systems; and alsoany of the class of nonimmune-type binding pairs, such as biotin/avidin;biotin/streptavidin; folic acid/folate binding protein; complementarynucleic acid segments; protein A or G/immunoglobulins; and binding pairswhich form covalent bonds, such as sulfhydryl reactive groups includingmaleimides and haloacetyl derivatives, and amine reactive groups such asisotriocyanates, succinimidyl esters and sulfonyl halides.

The term "activated binding intermediate" refers to the product obtainedfrom reaction of the binding substrate with the enzyme of the ADRC.

The term "deposited binding product" refers to an activated bindingintermediate which has been deposited onto a receptor.

The term "nucleic acid replication conjugate" refers to a conjugatecomprising a second member of a binding pair species, optionally aspacer, and a target nucleic acid sequence.

The term "nucleic acid replication binding pair complex" refers to thecomplex formed between the deposited binding product and the nucleicacid replication conjugate.

The term "replication control" refers to a mixture comprising areference nucleic acid sequence and a cognate set of primers which havebeen designed to facilitate nucleic acid replication of the referencesequence.

The term "reference nucleic acid sequence" or "reference sequence"refers to a template nucleic acid that is different from the targetnucleic acid sequence. The reference sequence may be incorporated withinthe ADRS and additionally replicated to serve as an assay control whichimproves quantitation.

The term "reference nucleic acid conjugate" refers to a conjugatecomprising a reference nucleic acid sequence, optionally a spacer, andthe analyte or analyte equivalent.

The term "signal-generating nucleic acids" refers to any nucleic acidwhich has been modified or labeled with a moiety capable of detectionvia enzymatic means or energy emission; including, but not limited to,fluorescent moieties, radioactive tags, or light-emitting moieties.

The term "primer" refers to a nucleic acid sequence that iscomplementary to a portion of at least one strand of the targetednucleic acid and whose purpose is to sponsor and direct nucleic acidreplication of the targeted sequence. Primers are designed to becomplementary to specific segments of the target or reference sequences,and may be used in combination with another primer, thus forming a"primer set" or "primer pair". Requirements for primer size, basesequence, complementarity and target interaction are discussed in theprimer section of the detailed description of the invention. The term"primer",as such, is used generally herein by Applicants to encompassany sequence-binding oligonucleotide which functions to initiate thenucleic acid replication process; such replication processes mayinclude, for example, PCR, LCR or other enzymatic reactions which employsingle rather than multiple oligonucleotide initiators.

The phrase "replicated nucleic acid sequences" or "replicated sequences"refers to the nucleic acid replication products produced within the ADRSassay scheme, and is used within this context to include both replicatedtarget sequences and replicated reference sequences.

The term "ligand" will refer to one member of an analyte specificbinding pair such as a molecule, protein, peptide, nucleic acid segment,therapeutic agents, polypeptide, toxin, nucleotide, carbohydrate, cell,microorganism, antibody, lectin, receptor, binding protein or chemicalagent that is either identical to or structurally related to an analyte,and is capable of binding to the second member of the analyte specificbinding pair. The ligand may be structurally modified to enable chemicalattachment.

The term "ligand reporter conjugate" refers to a conjugate comprising atarget nucleic acid sequence coupled to a ligand. The ligand may becoupled either at the 3' end, the 5' end or at any position between the3' and 5' ends of the target. Additionally, the ligand is capable ofcompeting with sample analytes for analyte binding sites on animmobilized capture reagent.

The term "immobilized analyte complex" refers to a complex formedbetween an analyte and an immobilized capture reagent.

The present invention provides an amplified detection method for thedetection and quantitation of an analyte in a sample. The methodcomprises, at step i) immobilizing an analyte to form what Applicantshave termed an "analyte-dependent reporter system" (ADRS). The ADRS willbe comprised of an immobilized target nucleic acid sequence which hasbeen immobilized in response to the presence of an analyte in thesample. Next, at step ii) the target nucleic acid sequence of the ADRSis contacted with a nucleic acid replication composition underconditions wherein the target sequence may be replicated. At step iii)the target sequences are replicated. Any of several known methods forreplication of nucleic acids may be employed. The replicationcomposition which is added will be comprised of the reagents necessaryfor replication of the target sequence. And finally, at step iv) thereplicated target nucleic acid sequences are detected, whereby thepresence of analyte may be determined. The replicated target nucleicacid sequences may be detected using any of a number of currentlyavailable reporter detection schemes; such as size differentiation,ligand capture, radioactive detection, luminescence detection,fluorophorescence detection, or any combination thereof; includingwherein any of these detection schemes may be enzymatically mediated.

Applicants' amplified detection method may be specifically designed tobe practiced in a number of different ways. For example, Applicants havepresented four possible variations of the method (see FIGS. 1, 2, 3, and6) which differ in the manner in which the target sequence is ultimatelyformed within the ADRS.

Multiple target nucleic acid reagents can be used simultaneously withinan assay for the detection of different analytes, or to provide assaycontrols. In a preferred embodiment of Applicants' method, referencenucleic acid sequences which are different from the target sequencewould be included within the ADRS at one or more steps and replicatedconcurrently with the target sequence under the assay conditions of thatmethod. These reference sequences would be designed to generatesequences which are detectably distinct from the replicated targetsequences, and would therefore serve as measures of internal control foreach particular assay methodology.

In another preferred embodiment, multiple target nucleic acid reagents,each specific for a separate analyte can be employed together in thesame assay milieu to facilitate simultaneous detection of multipleanalytes.

One embodiment of the present invention, termed the "Direct TargetDeposition Method", is illustrated in FIG. 1.

In Step a of this embodiment, the test sample containing the analyte (A)is first reacted with an immobilized capture reagent (B), such as anantibody, and then with a reporter conjugate comprising a target nucleicacid sequence (C) to form an analyte dependent reporter complex (ADRC)(D) from which excess reagents are removed by washing. In Step b, theADRC is contacted with a nucleic acid replication composition and areplication process is performed to produce replicated nucleic acids(E). In Step c, the replicated nucleic acids are detected. In thisembodiment, the reporter conjugate is a conjugate comprising a targetnucleic acid sequence and, for example, an antibody or other analytebinding reagent.

An obvious variation of the method, which is easily practiced by oneskilled in this art, is an adaption wherein after step a, any excess,nonimmobilized reporter conjugate remaining free in solution would beseparated from the immobilized capture reagent-analyte complex. Thisamount of excess, nonimmobilized reporter conjugate remaining free inthe analyte sample would be proportional to the amount of analyteinitially present in the sample. This nonimmobilized reporter conjugate,after separation from the bound analyte complexes, could then bereplicated while free in solution, for example, and the replicatednucleic acids are detected whereby the presence of analyte in the sampleis determined.

Another embodiment of the present invention, termed the "CatalyzedTarget Deposition Method", is illustrated in FIG. 2. In Step a of thisembodiment, the test sample containing the analyte (A) is first reactedwith an immobilized capture reagent (B), such as an antibody, and thenwith a reporter conjugate (C) to form an analyte dependent reportercomplex (D) from which excess reagents are removed by washing. In thisembodiment the reporter conjugate (C) is comprised of an enzyme capableof activating a moiety on the nucleic acid replication substrate (F)(such as horseradish peroxidase) and a member of a binding pair (such asan antibody). In Step b, the ADRC formed in Step a is reacted with anucleic acid replication substrate (F), which contains the targetnucleic acid sequence, to form an activated nucleic acid replicationintermediate (G), which deposits wherever receptor for the activatednucleic acid replication intermediate is immobilized to produce adeposited nucleic acid replication product (H). Excess reagents are thenwashed off. In Step c, the deposited nucleic acid Replication product iscontacted with a nucleic acid replication composition to producereplicated target sequence nucleic acids (E). In Step d, the replicatednucleic acids are detected.

Another embodiment of the present invention, termed the "CatalyzedIndirect Target Deposition Method",is illustrated in FIG. 3. In Step aof this embodiment, the test sample containing the analyte (A) is firstreacted with an immobilized capture reagent (B) (such as an antibody),and then with a reporter conjugate (C) to form an analyte-dependentreporter complex (D) from which excess reagents are removed by washing.In this embodiment the reporter conjugate (C) is comprised of an enzyme(such as horseradish peroxidase) which is capable of activating a moietyon the binding substrate (I). (I), the binding substrate, is a conjugatecomprised of this substrate and a member of a binding pair. In Step b,the ADRC is reacted with the binding substrate (I) to form an activatedbinding intermediate (J) which deposits wherever receptor for theactivated binding intermediate is immobilized, to produce a depositedbinding product (K). Excess reagents are then washed off. In Step c, thedeposited binding product is reacted with a nucleic acid replicationconjugate (L), which contains the target nucleic acid sequence and thesecond member of the binding pair, to produce a nucleic acid replicationbinding pair complex (M). Excess reagents are then washed off. In Stepd, the nucleic acid replication binding pair complex is contacted with anucleic acid replication composition to produce replicated targetsequence nucleic acids (E). In Step e, these nucleic acids are detected.

Another embodiment is a variation of the "Direct Target DepositionMethod" of FIG. 1, and is illustrated in FIG. 4. The object of thisembodiment is to provide a method for detecting several differentanalytes in a single sample, and may also be referred to as the"multianalyte method".

In FIG. 4, at Step a, the test sample containing different analytes (Aand A') is first reacted with the immobilized capture reagents (B andB'), and then with the reporter conjugates (C and C') each comprising atarget nucleic acid sequence to form the analyte dependent reportercomplexes (D and D') from which excess reagents are removed by washing.In Step b, the ADRCs are contacted with a nucleic acid replicationcomposition and a replication process is performed to produce replicatednucleic acids (E and E'). In Step c, the replicated nucleic acids aredetected. In this embodiment, there are more than one reporterconjugates, each comprising an analyte specific antibody or otheranalyte binding reagent linked to a target nucleic acid sequence. Thetarget nucleic acid sequence for each type of reporter conjugate has aspecific length, which is different in length from the target of anyother reporter conjugate. Replication of the target nucleic acidsequences thus gives amplification products of different lengths and thepresence of different analytes may be conveniently detected by analysisof the amplification products on the basis of size, such as in gelelectrophoresis. In a particularly preferred embodiment, the targetnucleic acid sequences which differ in length will be designed tocomprise the same 5' and 3' primer binding regions, so that the sameprimers can be used to replicate all of the various targets present inthe sample. In another preferred embodiment of multianalyte detectionwhich is useful for detection of multiple sequences in sample nucleicacids (the "multigene assay"), the reporter conjugates will be comprisedof target sequences which have been coupled to other nucleic acidsequences which are complementary to specific nucleic acids which may bepresent in the sample. The complementary sequences will hybridize tosequences present in the sample nucleic acids, thereby immobilizing thereporter conjugates. Target segments of the reporter conjugates willthen be replicated, indicating presence of the specific samplesequences.

An additional embodiment of the present invention, termed the"Competitive Binding Method", is illustrated in FIG. 6.

In FIG. 6, at Step a of this embodiment, the test sample containing theanalyte (A) is first reacted with an immobilized capture reagent (B),such as an antibody, and then with a ligand reporter conjugate (Q)comprising a target nucleic acid sequence bound to a ligand wherein theligand is capable of competing with the analyte (A) for binding sites onthe immobilized capture reagent (B). The reaction results in theformation of an immobilized analyte complex (N) leaving the ligandreporter conjugate (Q) unbound. The ligand reporter conjugate (Q) andthe immobilized analyte complex (N) are then separated by washing. InStep b, either the immobilized analyte complex (N) or the ligandreporter conjugate may be contacted with a nucleic acid replicationcomposition. In the case where the ligand reporter conjugate(Q) iscontacted, nucleic acid replication occurs and the presences of analyteis detected. In the case where the immobilized analyte complex (N) iscontacted, no replication occurs and no replicated nucleic acids (E) areproduced. In this embodiment, the ligand reporter conjugate is aconjugate comprising a target nucleic acid sequence and, for example, anantigen or other binding reagent capable of competing with the analytefor binding sites on the capture reagent.

Additionally, one of ordinary skill will recognize that the aboveseveral embodiments could be practiced employing alternativeimmobilization points throughout the assay. For example, in FIG. 6, theligand reporter conjugate could be immobilized and the capture reagentcould be free in solution.

Thus, in all of the above embodiments the production of replicatednucleic acids from a target nucleic acid sequence is used to amplifydetection of the analyte.

The process of the present invention may be used to detect the presenceof a wide variety of analytes. Generally, these include, but are notlimited to, plants, animals, nucleic acid segments, molecules, cells,microorganisms and fragments and products thereof, or any substance forwhich attachment sites, binding members or receptors (such asantibodies) can be developed. Of particular interest are pathogens,viruses and bacteria. It is contemplated that the sample material willbe a liquid, a gas or a solid to be dissolved in, extracted from orsuspended in a test fluid. The sample material will most likely be ofmedical, veterinary, environmental, nutritional or industrialsignificance. While not attempting to be limiting, it is contemplatedthat specimens for human, animal, or microbiological sources or habitatsmay be tested by the present method, including body fluids such asurine, blood, serum, plasma, milk, sputum, fecal matter, lung aspirates,exudates; microbial culture fluids; aerosols; crop materials; soils andground waters.

The immobilized capture reagent which binds the test analyte willgenerally be comprised of, for example, a binding protein, lectin,nucleic acid or an antibody, attached to an appropriate support. Anyknown antibody could serve as the antibody of the immobilized capturereagent. In addition, specific antibodies may be prepared and utilizedin this process. In certain instances analyte may be captured directlyby nonspecific interaction with the support, as in, for example, thehydrophobic interactions between proteins and polystyrene.

Suitable immobilization supports used in the ADRC, receptor supports andaffinity supports (to capture the replicated nucleic acids) includesynthetic polymer supports, such as polystyrene, polypropylene,polyglycidylmethacrylate, polystyrene, substituted polystyrene (e.g.,aminated or carboxylated polystyrene; polyacrylamides; polyamides;polyvinylchlorides, etc.); glass beads; agarose; or nitrocellulose, etc.These materials may be used as films, wells, beads, particles, pins,pegs or membranes. Alternatively, the supports could comprise magneticand non-magnetic particles.

These supports can be used to prepare different immobilized reagents.For example, depending on the approach and reagent configuration,separate immobilized support reagents could be prepared for binding theADRC, for binding of the activated conjugate receptor, or for capture ofthe products of nucleic acid replication during the detection step.Alternatively, under circumstances in which the analyte, receptor andproduct binding activities do not compete or interfere with the otherbinding functions, the analyte, conjugate and product binding reagentscould be co-immobilized on the same support. In this way the ADRC, andthe receptor support could be prepared and used as separate supports, orthe binding reagents could be combined on the same support. Analytebinding molecules, and receptors can be immobilized on the solid supportusing techniques well known to those skilled in the art. H. Weetall,Immobilized Enzymes. Antigens, Antibodies and Peptides, (1975) MarcellDekker, Inc., New York.

Typically, the immobilized capture reagent can be comprised of glycidylmethacrylate beads of about 30 u in diameter and an antibody such asgoat anti-Rabbit IgG antibody. Test beads and antibody are incubated at4° C. followed by a washing to remove excess antibody. The beads arethen treated with bovine serum albumin to bind any unreacted epoxidegroups and resuspended in buffer.

In practicing the present invention, two different types of reporterconjugates are contemplated by Applicants. The first type consists of atarget nucleic acid sequence coupled to an antibody or other bindingmember which recognizes an analyte. These can be prepared usingvariations of methods known to those skilled in the art for linkingproteins to amino-oligonucleotides, For example, this may beaccomplished using enzymatic tailing methods in which an amino-modifieddNTP is added onto the 3' end of the nucleic acid. A. Kumar, Anal.Biochem., 169, 376 (1988). Alternatively, amino-modified bases can besynthetically introduced into the nucleic acid base sequence. P. Li, etal., Nucleic Acids Res., 15, 5275 (1987). Antibodies can then beattached to amino-modified nucleic acids by substituting an antibody foran enzyme in the method of Urdea. M. S, Urdea, Nucleic Acids Res., 16,4937 (1988).

More specifically, preferred preparation of nucleic acid/antibodyconjugates involves the coupling of heterobifunctional cross-linkers tothe DNA oligonucleotide targets which in turn are coupled to antibodiesusing chemistry described by Tseng et. al. in U.S. Ser. No. 07/946247. Akey advantage of this linking chemistry over standard protocols in theart is that it reduces the occurrence of unwanted reactions such ashomo-DNA or homo-antibody polymers.

To facilitate the chemical attachment of the oligonucleotides to theantibodies, the oligonucleotides are amino-modified by introducing aprimary amine group at their 5' end during synthesis usingcyanoethyl-phosphoramidite chemistry. The amino-modifiedoligonucleotides are further modified with a hetero-bifunctional reagentthat introduces sulfhydryl groups. The reagent, N-succinimidylS-acetylthioacetate (SATA) is a heterobifunctional cross-linker agentthat uses the primary amine reactive group, N-hydroxyl-succinimide (NHS)to couple to the amino-modified oligonucleotides introducing anacetyl-protected sulfhydryl group. The antibodies are modified withanother NHS cross-linking agent, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC). The SMCC reacts with primary aminegroups within the peptides (e.g., the ε-groups on lysine) of theantibody, introducing a maleimide group (a free sulfhydryl reactivegroup) to the antibody. The maleimide-modified antibodies are mixed withthe SATA modified antibodies. The acetyl-protected sulfhydryl groups onthe SATA-modified oligonucleotides are activated with the addition ofhydroxylamine to produce reactive, free sulfhydryl groups (U.S. Ser. No.07/946247). The free sulfhydryl-containing oligonucleotides reactimmediately with maleimide-modified antibodies forming DNA to antibodyconjugates.

The second conjugate type comprises an enzyme coupled to an antibody orother member of a binding pair. These also may be prepared using methodswell known to those skilled in the art. D. G. Williams, J. Immun.Methods, 79, 261 (1984). Alternatively, enzyme-binding conjugates can begenerated using recombinant DNA and genetic engineering techniques. I.Pastan and D. Fitzgerald, Science, 254, 1173 (1991). Enzymes suitablefor use in a reporter conjugate include, but are not limited to,hydrolases, lyases, oxido-reductases, transferases, isomerases andligases. Others are peroxidase, glucose oxidase, phosphatase, esteraseand glycosidase. Specific examples include alkaline phosphatase,lipases, beta-galactosidase, horseradish peroxidase and porcine liveresterase. The choice of reporter conjugate depends upon which embodimentof the present invention is practiced.

Target Nucleic Acid

Logarithmic nucleic acid replication technology (for example, thepolymerase chain reaction (PCR), or the ligase chain reaction (LCR))provides highly sensitive means for amplifying copies of a specificnucleic acid sequence. These technologies afford two very importantcapabilities. One is the specificity of the replication process.Information from a single sequence can be specifically replicated in thepresence of samples containing complex mixtures of nucleic acids andhigh concentration of proteins. The second is the high sensitivityafforded by the process. Replications of target DNA on the order of >10⁶fold can be achieved by a temperature recycle process. Currently,pathogens can be detected in mixtures of unknown samples by sequenceprobes; however, the sensitivity approaches approximately only 10³cells/ml. Logarithmic sequence replication of target DNA has now greatlyextended probe test sensitivity enabling as few as 1 to 5 cells /100 mlto be detected, A. K. Bej et al., App. Environ. Microbiol., 56, 307(1990).

As noted above, the target nucleic acid is replicated to produceamplified copies of the target sequence nucleic acids. The design of thetarget sequence is important because replication requires suitablecomplementary primer(s); and also because the target can provide fordifferent means of detection and for flexibility in reaction conditions.

Specifically, the target nucleic acid sequence may vary in length from20 to 5000 bases. Preferably if the target is to be used for PCRamplification it will range between 30 and 1000 bases. If the target isused for LCR amplification the target length will range between 100 and500 bases. The target may be double-stranded (ds), comprising a hybridduplex of two complementary nucleic acid strands, or may alternatively,be single-stranded (ss). Double-stranded targets do not requireproduction of a complementary strand to participate in logarithmic chainpolymerization. Either or both strands can carry modified bases used forbinding or detection. When only one strand of the target is attached tothe support, the complementary strand will be free to anneal withprimers in the solution phase. Once removed from the support matrix,strand replication is unhindered. Double-stranded targets are thusparticularly useful for immobilized targets, since heat denaturationwill free one strand which is freed from the support for replication.

Single-strands of double stranded targets however, may also be used whenpreparing conjugate reagents. For example, as illustrated below, onlyone strand of the target is used during the first amplification cycle.Annealing and extension of primer #1 can then convert thesingle-stranded target to a double-stranded duplex. Acting in concert insubsequent cycles, primers #1 and #2 will lead to logarithmicreplication of the newly-synthesized double-stranded target nucleicacid. Single-stranded targets offer some reagent preparation advantagesin that they are 1) cheaper, since only one strand need be made, and 2)no prior annealing with the complementary strand is required. The sameprimers may be used for amplification of appropriately designed ss or dstargets.

In a preferred simplification of the Applicants' invention, logarithmicreplication can be achieved using a single-stranded target and a singleprimer. This is achieved by designing the target sequence to contain aprimer binding sequence at one end of the ss target and a complementsequence of the primer binding site at the opposite end of the targetstrand. Annealing and extension of the primer will result in theformation of a complementary target strand containing the identicalprimer binding sites. In this way both the (+) and (-) strands of theresulting ds target contain an identical primer site at opposite ends ofthe target duplex, and the same primer used in combination with thepolymerase and target nucleic acid promotes replication of both + and -target strands.

The single primer approach affords advantages in reduced assaycomplexity and increased reproducibility. Simplification is achievedsince only one primer must be prepared and provided for detection. Moreimportantly, the single primer can enhance productivity of the nucleicacid acid replication process since each primer has exactly the samemelting temperature (Tm). Temperature recycling constraints are thusmore easily controlled. Furthermore, the likelihood of nonspecificnucleic acid formation resulting from primer-dimer replication isreduced.

In other preferred embodiments, the base composition and sequence of thetarget nucleic acid sequence can be varied to accommodate differentassay requirements. For example, the target may contain sequencesegments which are not amplified during replication, or variable regionsused to alter the length of the target sequence. It is contemplated, forexample, that a target nucleic acid sequence may be designed to containa coupling linkage for the attachment of an antibody or ligand at the 5'end and a primer binding site at the 3' end with a variable regioninserted between. The variable region could be of any length orcomposition, limited only by the requirements of the targetamplification method. This is illustrated in FIG. 5, which provides anexample of a reporter conjugate wherein the target nucleic acid has beenconjugated to an antibody through a chemical coupling linkage at the 5'end. The target oligonucleotide may contain a 5' binding regioncomplementary to one of the replication primers, and a 3' site forbinding the other replicated primer. Within the target sequence is avariable region of nucleic acid bases, which could be variable in lengthor in sequence, thereby providing alternative means of detection of thereplicated targets based on size or other factors, such as ability tobind, or ability to emit distinctive signals (such as fluorescence,radioactivity, etc.).

Another preferred embodiment for multi-analyte detection is shown inFIG. 4, wherein target nucleic acids which vary in length are used todetect various analytes. For example, a series of nucleic acid targetshaving the same primer binding sites and same sequence, but differingonly in length of the inner target region could be prepared, and coupledto different binding pairs which are capable of binding to the differentanalytes (such as analyte-specific antibodies, lectins, receptors,etc.). The replication products of each of these receptor conjugatescould then be readily distinguished on the basis of size, andconveniently visualized, for example, by gel electrophoresis. In a"multigene assay", where the analytes to be detected are specificsequences of RNA or DNA contained within sample nucleic acids, thereporter conjugates are comprised of targets of varying lengths coupledto nucleic acid sequences which are complementary to specific portionsof the sample nucleic acid. In this way, multiple genes or sequencesites within one sample can be conveniently screened in one assay. Thehigh resolution capability of nucleic acid separation, wherein sequenceswhich vary in length by only one base can be resolved, renders thismethod extremely attractive when large numbers of samples containingmultiple analytes are to be screened.

Additionally, the target sequence can be designed to facilitatedetection of the amplified nucleic acids which are capable of emittingdetectable signals. For example, the target sequence may provide forincorporation of labeled primers or labeled bases (e.g., fluorescence,radioactive, light emitting) to produce the correspondingly labeledsignal generating nucleic acids. The type, number of labeled bases andposition within a chain, and between complementary chains, may bedesigned to facilitate signal detection. In a preferred embodiment, itis desired to position specifically labeled bases in the sequence so asto enable energy transfer between fluorophores, or to enable enzymechanneling between proximally positioned coupled enzymes.

Specifically, energy transfer between suitably labeled bases can beachieved if the distance between the excitation fluorophore (F1) and theemission fluorophore (F2) are within 12 bases (ca. 50A°) in the helicalduplex assemblage R. A. Cardullo et al., Proc. Natl. Acad. Sci. USA, 85,8790 (1988). A more preferred distance is between 5 to 12 bases. Thiscan be achieved by designing the target and primer sequences so that oneof the labeled bases (F1 and F2) is alternately incorporated in thesignal nucleic acid at each turn of the helix. Alternatively, the basesequence of the target or primers can be designed so that F1 and F2 areincorporated into opposite strands of the signal nucleic acid. Theposition of labeled bases is controlled so that on strand hybridization,F1 and F2 are positioned within the duplex at a distance of <50A°. Morepreferable, F1 and F2 will be positioned on the same side of duplex oneturn apart. Thus, within the signal nucleic acid, both interchain andintrachain labeled bases can position the fluorophores within a distancesuitable for energy transfer.

The requirements of fluorophores which participate in energy transferare well documented. L. E. Morrison, Anal. Biochem., 174, 101 (1988).Generally, to achieve energy transfer it is also important to select theappropriate combination of fluorophores used for labeling the excitation(F1) and emission (F2) bases so that the emission spectrum of theexcitation fluorophore (F1) overlaps with the adsorption or excitationspectrum of the excitation fluorophore (F2). For example, the followingfluorophore combinations include commonly available suitable candidatesfor energy transfer:

    ______________________________________                                        Excitation Fluorophore (F1)                                                                      Emission Fluorophore (F2)                                  ______________________________________                                        Pyrenebutyrate     β-Phycoerythrin                                         Fluorescein Texas Red                                                         Lucifer Yellow Rhodamine                                                      Lucifer Yellow Texas Red                                                      Fluorescein Rhodamine                                                         Fluorescamine Fluorescein                                                   ______________________________________                                    

In another preferred embodiment, the sequences of the target and primerscan be designed to incorporate bases labeled with the first member of abinding pair (e.g. digoxigenin, biotin). The incorporated labeled basescan be used to either immobilize the resulting nucleic acids, or tocomplex them with a second member of the binding pair labeled with areporter (e.g. streptavidin-alkaline phosphatase,antidigoxigenin-alkaline phosphatase). It is contemplated that thetarget sequence may be designed to enable the incorporation of differentbases or primers; one or more labeled with binding members (e.g.,biotin); and one or more labeled with a reporter(s). It is desirable tocontrol the sequence so that the biotin-labeled bases are incorporatedpredominantly at one end of a chain and the reporter bases incorporatedat the other end or some distance from the binding members.Nevertheless, in designing the base sequence it is important to avoidboth consecutive runs of C's and G's (3 or more) at the 3' ends, as wellas with palindromic sequences. For example, a target gene sequence couldcontain the following sequence:

       1                                                        45                  5' ATG CGT AGC AGC TTT ACC GCA GAG ATC ATG CCT ATG TAC CAT GCT 3'            3' TAC GCA TCG TCG AAA TGG CGT CTC TAG TAC GGA TAC ATG GTA CGA 5'              -    46                                   75                                 5' ATC CTA CCT GTA AGT CAT AGC TGT TTC CTG 3' SEQ ID NO:1                     3' TAG GAT GGA CAT TCA GTA TCG ACA AAG GAC 5' SEQ ID NO:2                

The nucleic acid replication substrates are comprised of a targetnucleotide sequence, optionally a spacer, and a moiety capable of beingactivated by an enzyme. The target nucleic acid sequence is preparedaccording to the guidelines set out above. The moiety capable ofactivation by an enzyme may be any moiety which forms a reactiveintermediate which can bind to a receptor on the solid support of theimmobilized capture reagent. In preferred embodiments the enzymereactive moiety is tyramine.

Reporter conjugates, including the nucleic acid replication substrateand nucleic acid replication conjugate, may contain a molecular spacersegment linking the two functional elements of the conjugate. Onepurpose of the spacer is to extend the replication segment of the targetor binding functions away from the surface of the solid phase support.Useful spacers are well known in the affinity chromatography art. Forexample, H. Schoot, Affinity Chromatograph, (1984), Marcell Deckker,Inc., New York, describes different spacers and their use.Advantageously, the spacer includes a chain of up to about 50 atoms,preferably 5 to 30 atoms. In composition, spacers may be apolyfunctional segment including, but not limited to, one or more of thegroups: peptide, hydrocarbon, polyalcohol, polyether, polyamine,polyimine and carbohydrate e.g. -glycyl-glycyl-glycyl- or otheroligopeptide, carbonyl dipeptide, and omegaaminoalkane-carbonyl radicalsuch as --NH--(CH₂)₂ --CO--, a spermine or spermidine radical,omega-alkanediamine radical such as --NH--(CH₂)₆ --NH-- or --HN--CH₂--CH₂ --NH--. The spacer segment may also be comprised of polymericunits such as polysaccharide, polyethylene oxide radicals, glyceryl,pentaerythritol and like radicals. The spacer segment may be linkeddirectly or linked through a divalent heterobifunctional orhomobifunctional couplers, for example SATA (N-succinimidylS-acetylthioacetate), SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate), p-phenyl diisothiocyanate, dithiobissuccinimidyl propionate, 1,4-butanediol diglycidyl ether, adiisocyanate, carbodiimide, glyoxal, glutaraldehyde or sulfosuccinimidyl6-(4'-azido-2'nitro-phenylamino)-hexanoate.

The length of the target nucleic acid sequences may be extended beyondthe sites of primer attachment. The extended length of the target thuscan provide an alternative spacers and thus reduce the length oreliminate the need for a molecular spacer, and also perhaps increase theefficiency of target replication. For example, bases added at the targetattachment site will extend the target segment away from the point ofimmobilization. In this way, spacer length can be reduced or in someinstances eliminated. Generally, adding 5 to 30 bases to the target willbe sufficient to increase the efficiency of immobilized targetreplication. The composition and length of molecular spacers aredesigned to prevent interference during amplification of nucleotidetarget sequence. Recent findings indicate that target sequences next tothe aminolink spacer are accessible for primer attachment andlogarithmic chain reaction. (S. Stamm and J. Brosius, Nucleic AcidsResearch, 19 1350 (1991).

Once the target nucleic acid sequence is designed, the nucleic acidreplication substrates may be prepared using well-established proceduresdeveloped for preparation of enzyme-labeled oligonucleotide probes. Seee.g., G. H. Keller and Manak, M. M., DNA Probes, (1989), pp. 136-148,Stockton Press, New York. More specifically, during the synthesis of thetarget nucleic acid sequence, a base modified with a spacer armcontaining a primary amine can be introduced at either the 5' end or the3' end of the target. Reagents for introducing a base containing a 5'amino group are commercially available (C8-aminohexyl-ATP andN6-amino-hexyl ATP, Sigma Co)), and methods of accomplishing theintroduction into the sequence are known in the art. TheN-Monomethoxytrityl-C6-AminoModified cyanoethyl phosphoramidite reagent(Clontech Laboratories Inc., 4030 Fabian Way, Palo Alto, Calif. 94303)or Aminolink™ 2 (Applied Biosystems, Inc.) provide an easy means ofintroducing a 5' terminal primary aliphatic amine to an oligonucleotideduring synthesis of a target oligonucleotide. Detailed procedures forthe coupling reaction are available from Clontech bulletin no.PB022789-1, or from Applied Biosystems, Inc., Model 392 Manual. Once theamino-modified nucleic acid target has been prepared, it can then bereacted with succinic anhydride; which extends the length of the sidechain and also provides a terminal carboxylic acid which can beactivated using standard methods to form the N-hydroxysuccinimide (NHS)intermediate (1). This intermediate can then be chemically coupled totyramine, for example, to form a nucleic acid replication substrate (2).

(1) NHS(C)n linker-5' target 3'

(2) Tyramine-(C)n linker- 5' target 3'

Generic reagents are also available from Cruachem (460 Spring Park,Herndon, Va. 22070) Clontech Laboratory (4030 Fabian Way, Palo Alto,Calif. 44303) or Applied Biosystems, Inc. (Foster City, Calif.) forintroducing internal single or multiple amino groups into the genesequence. However, end-labeled oligomers tend to be more accessible forbinding and reaction than internally labeled nucleic acids.

In another preferred embodiment, it is contemplated that the sequencesof the target nucleic acid can be designed to incorporate a ligand. Asused herein the term ligand will encompass both ligands which arestructurally related to the analyte, and ligands which mimic analytebinding, so long as the ligands have the ability to compete with ananalyte for receptor binding sites. Thus, ligand-target conjugates mayfunction as a first member of a binding pair and can mimic the bindingproperties of an analyte, competing with the binding of an analyte to asecond member of a binding pair (e.g., an antibody). For the purposes ofthe present invention ligands of less than 3000 molecular weight arepreferred whereas ligands with molecular weights of less than 1500 aremost preferred.

Variability in the positional orientation and number of the incorporatedligands lends flexibility to the target design, allowing for increasedassay sensitivity and optimized conjugate interaction with the capturereagents. It is contemplated that ligands can be incorporated into oneor both strands of a duplex target nucleic acid. Positionally, ligandscan be incorporated either at the 5' or 3' ends of the target orincorporated on internal bases within the nucleic acid sequence, whereincorporation at the ends is generally preferred. It is contemplatedthat any number of ligands may be incorporated per target, however,where the object is to achieve maximum sensitivity of the assay, arelatively small number of ligands is preferred, where a range of one totwo is most preferred. In the situation where maximum rate of conjugatecapture is desired, a high number of ligands per target is preferred.

The method of incorporation of the ligand into the nucleic acidsequences may be accomplished either by chemical or enzymatic means, orby direct incorporation of ligand labeled bases into the targetsequence. Chemical incorporation would utilize chemistry similar to thatused for the synthesis of the tyramine replication substrate, aspreviously discussed. Typically, a base modified with a spacer armcontaining a primary amine can be introduced at either the 5' end or the3' end of the target which can be further modified to aN-hydroxysuccinimide (NHS) intermediate which may in turn be chemicallycoupled to the ligand.

In a preferred approach, ligand-incorporated sequences are preparedusing ligand-labeled bases or primers during polymerase chain reaction.It is contemplated that ligand labeling can be accomplished eitherthrough the incorporation of primers modified with ligand(s) or by usingligand-labeled dNTPs. Ligand labeled primers can be prepared usingstandard oligonucleotide cyanoethyl phosphoramidite chemistry bysubstituting selected bases with ligand-modified phosphoramidite basesduring primer synthesis. Alternatively, if primers are prepared withmodified bases containing a linkable molecular spacer, the ligands canbe chemically linked to the spacer after primer synthesis. Anothermethod would make use of ligand-labeled dNTPs or amino-modified dNTPswhich can be incorporated into a target nucleic acid sequence during theamplification procedure.

There are several advantages to synthesis of ligand-incorporated nucleicacid sequences by PCR as opposed to chemical or enzymatic means. Forexample, because of failure of sequences inherent in chemical synthesis,targets of longer than 100 bases are more easily constructed.Additionally, where labeled primers are used, it is possible to controlboth the positioning and number of ligands within one or both strands ofthe target sequence by the appropriate placement of the ligand in theprimers.

In contrast, although the use of labeled dNTPs facilitates thepreparation of multivalent ligand reporter conjugates, precise controlover ligand number and labeling pattern is less reliable. This isbecause dNTP-ligand incorporation is dependant on both polymerasediscrimination between dNTP and modified dNTP analogs and the frequencyof occurrence of a specific base in the target nucleic acid sequence.

It should be appreciated that the above discussion regarding preparationof target nucleic acid sequences and replication conjugates is equallyapplicable to preparation and design of the reference nucleic acidsequences and reference replication conjugates of the invention.

The binding substrate is comprised of a first member of a binding pairspecies, optionally a spacer, and a moiety capable of activation by anenzyme. The spacer and moiety are the same as those described for thenucleic acid replication substrate. Members of specific binding pairssuitable for use in practicing the invention can be of the immune ornon-immune type. Immune-specific binding pairs are exemplified byantigen/antibody systems or hapten/anti-hapten systems. The antibodymember, whether polyclonal, monoclonal or an immunoreactive fragmentthereof, of the binding pair can be produced by customary methodsfamiliar to those skilled in the art. The terms immunoreactive antibodyfragment or immunoreactive fragment refer to fragments which contain thebinding region of the antibody. Such fragments may be Fab-type fragmentswhich are defined as fragments devoid of the Fc portion, e.g., Fab, Fab'and F(ab')₂ fragments, or may be "half-molecule" fragments obtained byreductive cleavage of the disulfide bonds connecting the heavy chaincomponents of the intact antibody. If the antigen member of the specificbinding pair is not immunogenic e.g., a hapten, it can be covalentlycoupled to a carrier protein to render it immunogenic.

Non-immune binding pairs include systems wherein the two componentsshare a natural affinity for each other but are not antibodies.Exemplary non-immune binding pairs are biotin-avidin orbiotin-streptavidin, folic acid-folate binding protein, complementaryprobe nucleic acids, Proteins A, G, and immunoglobulins, etc. Alsoincluded are non-immune binding pairs which form a covalent bond witheach other. Exemplary covalent binding pairs include sulfhydryl reactivegroups such as maleimides and haloacetyl derivatives and amine reactivegroups such as isothiocyanates, succinimidyl esters and sulfonylhalides, etc. In preferred embodiments, an exemplary binding substratewould be a conjugate of biotin coupled to tyramine via aN-hydroxysuccinimido linker molecule. The binding substrate can besynthesized using well known methods. M. N. Bobrow, et. al., J. Immunol.Methods, 125, 279, (1989)

The nucleic acid replication conjugate is comprised of a second memberof a binding pair species, optionally a spacer, and a target nucleicacid sequence. The target nucleic acid sequence and the spacer aredesigned and prepared according to the principles set out above. Thesecond member of the binding pair species is chosen so as to becomplementary to the first member of the binding pair species utilizedin the binding substrate. As noted above, in a preferred embodiment thebinding substrate is a conjugate comprised of biotin, a spacer andtyramine. Thus, the choice of the second member of the binding pairspecies used in the nucleic acid replication conjugate will be avidin orstreptavidin, and thus the binding substrate will comprise avidin orstreptavidin as the second member of the binding pair species. Thenucleic acid replication conjugates can be prepared using wellestablished procedures. See, e.g., G. H. Keller and Manak, M. M., DNAProbes, (1989), pp. 136-148, Stockton Press, New York. More specificallyduring the synthesis of the target oligonucleotide, a base modified witha spacer arm containing a primary amine could be introduced at eitherthe 5' end or the 3' end. Reagents for introducing a 5' amino group arecommercially available (e.g. Aminolink™ 2; Applied Biosystems Inc., 800Lincoln Centre Drive, Foster City, Calif. 94404). Aminolink™ 2 is addedas the last step in the synthesis of the oligomer. The amino-modifiednucleic acid target is then activated by a bifunctional ester to bothextend the length of the side chain and to provide a terminal carboxylicacid which can then be activated using standard methods to formN-hydroxysuccinimide (NHS) intermediate (1). This intermediate can thenbe chemically coupled with avidin to form the nucleic acid replicatingconjugate (3):

(1) NHS(C)n linker-5' xxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 3'

(3) Avidin -(C)n. linker- 5'xxxxxxxxxxxxxxxxxxxxxxxxxxxxxx 3'

Generic reagents are also available from Cruachem, and Clontech forintroducing internal single or multiple amino groups into the targetnucleic acid sequence. However, end labeled oligomers tend to be moreaccessible for binding and reaction than internally labeled nucleicacids.

Primers

In current practice, replication of the target nucleic acid sequencerequires "primer" oligonucleotides which, as used herein, refers to alloligonucleotides which anneal to target sequences to facilitatereplication of the target.

When target replication is performed by polymerase chain reaction twospecific primers are used. Each primer specifically hybridizes with oneof the two complementary strands of the target (or if the target issingle-stranded (ss) one of the primers is specific for the secondstrand after synthesis). Replication of the target requires that the 5'end of the primer which is complementary to the (-) sense target strand(primer #2), corresponds to a region of the (+) sense strand which is 5'to the 3' end of the (+) sense strand specific primer (#1).Additionally, the primers should not contain regions with sufficientcomplementarity to form primer-dimers. Within these constraints, thetotal length of the primers may range from shorter than, to longer than,the target. In general, primers 10-30 bases in length are mostpractical.

    ------ 5' primer #1       <                                                       + sense 5' -XXXXXXXXXXXXXXXXXXXXXXXXXXX-3'                                    - sense 3' -YYYYYYYYYYYYYYYYYYYYYYYYYYY-5'                                 primer #2  5'  ----------->                                              

Primers may also contain sequences at their 5' ends that have nocomplement in the target (5' overhang or 5' mismatch).

    ----------5' primer #1    <                                                     - + sense 5' -XXXXXXXXXXXXXXXXXXXXXXXXXXX-3'                                  -      5' ----------->     primer #2                                          - - sense 3' -YYYYYYYYYYYYYYYYYYYYYYYYYY-5'                                   - (X, Y represent complemetary bases.)                                  

This 5' overhang or 5' mismatch can be used to incorporatefunctionalized bases (e.g., signal-generating or bindingmember-derivatized bases) on the primers, or to extend the length of thereplicated nucleic acid products by adding extra sequences. Theseadditions can be useful for capture of the resulting nucleic acidsand/or signal detection. In the primer a 3' segment complementary to thetarget can be joined to a variety of different 5' segments. Thus, aseries of primers with a fixed hybridizing region linked to differentsignal generating tails can be made. The signal generated would dependon the 5' region of the primer(s) used and could be tailored to thedetection method of interest. Hence, a single target sequence can beused with different primers containing varying 51 overhangs ormismatches to generate a number of different sequence specificresponses.

Typically, in PCR-type amplification techniques the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Some simple rules are useful in selection anddesign of the primers. Typically, primers should be 10 to 35 base pairsin length having a 50 to 60%, G+C composition. The calculated Tm's for agiven primer pair should be balanced. For this purpose, a 2° C. for A orT and 4° C. for G or C can be added together to estimate the Tm of theoligonucleotide. (Thein and Wallace, "The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorderst",inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986)pp. 33-50 IRL Press, Herndon, Va.). Depending on the selectedconditions, Tm's between 55° C. and 80° C. are suitable. In addition tothe Tm's, the complementarity at the 3' end of the primers is animportant consideration. Generally, complementarity of primer pairsshould be avoided, especially at the 3' ends. Also, consecutive runs ofC's and G's (3 or more) at the 3' ends of the primers along withpalindromic sequences should be avoided. Consideration should also begiven to the concentration of primer molecules in the replicationmilieu. Primer concentrations between 0.01 and 1.0 uM are generallysuitable, with concentrations of about 0.05 to 1.0 uM being optimal.

When the ligase chain reaction (LCR) is used for replication of a targetdouble-stranded nucleic acid, two sets of target-specific primers willbe required. The members of one set of primers are complementary toadjacent sequences found on a given strand of the target, while themembers of the second set are complementary to adjacent sequences on theopposite strand. In this way a set of adjacent primers is specific foreach target strand. During the replication process the target nucleicacid is heated to denature the two target strands. The fourcomplementary oligonucleotide primers comprising the two primer sets arethen hydridized near their melting temperature to the separated targetstrands. A thermal-stable ligase will covalently attach the adjacentprimers on each target strand. Only adjacent primers that are perfectlycomplementary to the target will be ligated together. In this way, theproducts from the first stage of ligation become targets for the nextround of ligation. The products thus increase exponentially withcontinued cycles of target denaturation, primer hydridization andligation steps.

The requirements for non-complementarity between primers, size, basecomposition and melting temperature requirements of the primers tend tobe similar to those stated above for PCR replication. Generally, primersfor LCR replication should be sufficiently long so that each willpreferentially bind to its specific binding site on the target nucleicacid. To insure specificity of ligation, reactions can be carried outnear the melting temperature (Tm) of the oligonucleotide primers. Athigher temperatures single-base mismatch at the junction can form. Thisresults not only in an imperfect double helix but destabilizeshydridization of the mismatched oligonucleotides.

In either PCR or LCR type replications, the primers may contain baseslabeled with reporter(s) or labeled with one member of a specificbinding pair. For example, biotin and fluorescein residues may beincorporated into the primer during CE phosphoramidite synthesis (NENProducts, Du Pont, Boston, Mass.; or Clontech). Incorporation of theprimers during amplification will also result in nucleic acid productscontaining biotin and fluorescein. In this way, primer incorporationduring replication process can be used as a preferred means ofintroducing reporters and affinity labels in the replicated nucleicacids.

Assay conditions

Practicing the analyte-dependent reporter system of the presentinvention requires several steps. First, an analyte-dependent reportercomplex (ADRC) is formed. This is accomplished using well-knownimmunoassay reagents and techniques. The reagents can be configured forsequential, competitive, sandwich, and immunometric immunoassayapproaches. Harlow, E. and Lane, D. L., Antibodies--A Laboratory Manual,(1988), pp. 555-612, Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.

Once the ADRC complex has been formed excess free reagents is removed.This is an important step since any free reagent and non-specificallybound reporter can contribute to the replication process. To aid inreducing non-specific binding, stringent wash conditions which do notcause dissociation of the ADRC, such as used in nucleic acidhybridization tests, can be employed since target replication andreadout are based on nucleic acid chemistry. For example, heating, pHchanges, or (and) the addition of formamide, detergents and salts can beused to increase the efficiency of the wash step. Too stringentconditions can lead to dissociation of the ADRC or destruction of theimmunoassay reporter. Tolerance to stringent wash conditions will varywith the nature of the analyte, binding member and specific reporterused. The stringent conditions must, therefore, be experimentallyoptimized for each assay. However, in washing to reduce non-specificbinding, if some of the ADRC is lost, this can be compensated for byadditional target replication realized by increasing the number oftemperature recycle steps.

In cases where the ADRC is sensitive to stringent wash conditions, acatalyzed reporter assay (FIG. 2) can be used. In this configurationreaction of the ADRC with the substrate results in covalent couplingbetween the target nucleic acid and the support. After coupling, thesolid support is washed leaving the covalently bound target nucleic acidcomplex. Specifically, a tyramine target substrate could be used with anHRP enzyme reporter to covalently couple the target to the support. Inthis way, enzyme amplifications could be accomplished and stringent washconditions used before target replication.

In the next step, the target nucleic acid sequence is replicated. If PCRis the replication method used, the target sequence is mixed with thenucleic acid replication composition (100 ul) comprising two primers(100 pmol/primer), a thermally stable DNA polymerase such as Tag DNApolymerase (2 units), required nucleotides (dNTP 200 uM/base) which maybe signal generating or ligand-containing nucleic acids, Tween 20detergent (0.05%), 20 mM TRIS/HCl buffer (pH 8.3), MgCl₂ (1.5 mM), KCl(25 mM), and nuclease-free gelatin (100 ug/ml).

Generally, excess Mg⁺⁺ in the replication reagent composition can resultin the non-specific amplification whereas insufficient Mg⁺⁺ will reduceyields. It is known that deoxynucleotide triphosphates (dNTP) bind Mg⁺⁺,and the amount of binding depends upon the dNTP concentration. In areaction composition containing all four bases (dNTP) this leaves afinal free Mg⁺⁺ concentration of ca. 0.7 mM of the original 1.5 mM Mg⁺⁺.If the dNTP concentrations are changed significantly, a compensatorychange in MgCl₂ may be necessary.

Because of the diversity of applications in which the present inventioncan be used, adjustments in the concentrations and reagent compositionsof the nucleic acid replication reagent composition may be required.Practical guidelines for optimizing and adjusting the replication milieucan be found R. K. Saiki and Gelfand, D. H., in PCR Technology, (1989),pp. 7-22, Stockton Press, New York.

The reaction mixture is covered with mineral oil to prevent evaporation.The target nucleic acid is then denatured to separate the duplex nucleicacid strands. Generally this is carried out a high temperature (90-95°C.) for 15 seconds; however, a longer initial time can be required toassure complete denaturation. Annealing of the oligonucleotide primersto the target nucleic acid template is usually accomplished by loweringthe temperature to 37 to 60° C. for 30-60 seconds. Polymerase extensionof the primers can then be accomplished by equilibration at 72° C. for10 to 60 seconds, depending on the length of the signal-generationproducts. The denaturation, primer annealing and primer extension stepsare repetitively carried out in sequence to amplify the strand number ofthe signal-generation product. The temperature cycling is typicallyperformed 10 to 40 times depending on the desired degree of replication.The reaction can then be stopped by addition of EDTA (10 mM) andchilling to 4° C.

In general, if LCR is the replication protocol used, the target sequenceof the ADRC is mixed with two sets of adjacent oligonucleotides (40 fmoleach); each set will be complementary to one of the complementary targetnucleic acid strands; in 10 ul of buffer containing 20 mM Tris.HClbuffer pH 7.6, 100 mM KCl, 1 mM EDTA, 10 mM NAD, 10 mM dithiothreitol, 4ug salmon sperm DNA and 15 nick-closing units of a thermostable ligase,for example, T. aquatics ligase, (Tabor, S. and Richardson, D. C. (1985)Proc. Natl. Acad. Sci. USA 82, 1074-1078).

The reaction mixture is then protected from evaporation, for example, bycovering with a drop of mineral oil, and then heated to 94° C. to insureseparation of the target strands. Annealing of the oligonucleotideprimers to the target is usually accomplished by lowering thetemperature to between 37 to 60 degrees. An optimum temperature close tomelting temperature of the primers is usually selected, but must bedetermined dependent upon the primer length and composition of thespecific primers. The melting and primer annealing cycle are thenrepeated 10 to 30 times. The reaction is then chilled to 4° C. to stopthe reaction.

The next step involves detection or visualization of the amplifiednucleic acids. This can be accomplished by several means including (a)direct detection of the duplex nucleic acids using intercalating dyes;(b) indirect or direct detection of ligands, isotopes or reportersincorporated in the nucleic acids; (c) hybridization of reporter probesto the amplified nucleic acids; or (d) direct detection of replicatedproduct following separation of replicated product from reaction milieubased on increased size of replication product.

Specifically, amplified nucleic acids can be detected in the reactionmixture by adding intercalating dyes. Of particular use are those dyesof the ethidium, phenazines, furocomarins, phenothiasines and quinolinetype which on intercalation with the duplex strands of nucleic acidschange dye detection properties. General reviews and further informationcan be obtained in Berman et al., Ann. Rev. Biophys. Bioeng., 20, 87(1981). For example, a preferred dye is ethidium bromide which onnucleic acid intercalation can be detected by excitation of the reactionmixture with short-wave uv light (259 nm).

Incorporation of modified free bases or modified primers during nucleicacid replication provides a means of introducing bases modified withligands, isotopes, or reporters. If ligase-type replication is used,oligonucleotides with modified bases already incorporated could be usedto replicate the target sequences. These techniques afford severaldetection strategies. For example, the incorporation of biotinylated orligand modified bases provides means of isolating the amplified nucleicacid products from solution onto a solid support and discarding theunincorporated bases. The addition of an avidin-signal-generatingcorrugate there facilitates detection. The amplified sequences may alsocontain signal-generating labeled bases. These can be detected directlyon the solid phase support. Alternatively, methods of collecting anddetecting biotinylated DNA fragments on magnetic beads containingimmobilized avidin or streptavidan are described by J. Wahlberg et al.,Mol. Cell Probes, 4 285 (1990).

In another alternative, the sequence of the amplified segment could bedesigned to position fluorescent bases within the signal nucleic acidsfor energy transfer or position the biotinylated bases so that bindingof avidin-labeled enzyme(s) reporters would result in enzyme channeling.Using these approaches the amplified target can be detected without theneed for separation from the unincorporated bases. According tomolecular modeling and recent reports, R. A. Cardullo et. al., Proc.Natl. Acad. Sci. USA, 85, 8790 (1988), energy transfer can be achievedat distances between the fluorophores of as much as 12 bases apart.However, optimum distance appears to be somewhere between 5 to 12 bases.At one fluorophore base per helix turn, this positions the donor andacceptor fluorophores in appropriate proximity for energy transfer.

Analyte Quantitation

The analyte-dependent reporter system (ADRS) response depends upon thequantity of analyte present in the sample, and also upon the efficiencyof sequence amplification. While analyte concentrations in samples canbe interpolated from standard curves by experimentally relating assayresponse under fixed reaction conditions and known analyteconcentrations, the efficiency of sequence replication is difficult topredict and control because of procedural and reaction variables.Furthermore, amplification is highly sensitive. Hence, if false negativetest response can be identified, the absence of analyte can be morereliably determined and the useful range of the assay extended.

Applicants contemplate that at least two types of internal controls canbe used to compensate for changes in the efficiency of sequencereplication and to provide means of identifying false negative testresponse. Applicants refer to these as "amplification, or replicationcontrol", and "capture control". An "amplification control" refers to anucleic acid sequence called the "reference sequence" (specifically, adifferent sequence than the target sequence nucleic acid) and itscorresponding cognate primer(s). In use, the reference sequence andcognate primers could be included in the nucleic acid replicationreagent composition, and would serve to demonstrate that reactionconditions are permissive for sequence amplification. During testing ifthe amplification control yields a signal, but the target sequence doesnot, then the lack of target sequence amplification cannot be the resultof test conditions non-permissive for amplification and the resultindicates an analyte concentration below the detectable level.

The amplification control can also serve as the internal reference foranalyte quantification. For this application, the reference sequence isadded at a known concentration approximating the analyte concentrationto be detected. During the replication reaction, the reference sequencewill theoretically be amplified with the same efficiency as the targetsequence. By determining the ratio of the signal responses from thereference sequence and the target sequence, the concentration of theanalyte may be determined. In this approach standard curves aredetermined by measuring the ratio of the responses resulting from thetarget sequence and the reference sequence in samples containing a rangeof known analyte concentrations and a fixed concentration of referencesequence. In this way, variations in efficiency of nucleic acidreplication can be compensated. Assay response can thus be moreaccurately related to analyte concentration.

The reference sequence must be similar in molecular weight to theamplification sequence, but must be capable of producing a separate anddistinct "replicated reference nucleic acid" which is detectablydistinguishable from the replicated target sequence nucleic acid. Thiscan be achieved by designing target and reference sequences to containunique bases. In this way during replication, a unique base or primerlabeled with ligands, reporters, isotopes or reactive groups can beincorporated respectively into the nucleic acid products of both thereference sequence nucleic acids and target sequence nucleic acids. Theresulting nucleic acids are thus labeled with separate reporters, or canbe isolated for detection via hybridization reactions or binding withcomplementary members of a specific ligand binding pair.

A "capture control" comprises an analyte reference sequence conjugate(properly configured for each embodiment--see below) and cognateprimer(s). The capture control sequence conjugate would be includedduring the analyte capture step of the assay; and the cognate primerpair would be included in the nucleic acid replication composition. Inthis configuration, a failure to detect the capture control replicationscould result from either a lack of capture or conditions non-permissivefor amplification. By using different sequences for the capture andamplification controls, both controls can be included in each reactionchamber allowing differentiation between failed capture and failedreplication reaction conditions. Inclusion of these controls is possibleonly because each of the three amplification sites, (target sequence,amplification control and capture control) could be designed to generatea sequence-specific, differentially detectable signal.

dNTP and primer levels should be adjusted so that each of the threereplication reactions can proceed to completion. Targets and cognateprimers should have similar Tm, length and other characteristics thateffect amplification efficiency.

EXAMPLES

The following examples are meant to illustrate key embodiments of theinvention but should not be construed to be limiting in any way.

Preparation of Reagents

Oligonucleotides to be used as the reporter or as primers are preparedusing standard cyanoethyl (CE) phosphoramidite coupling chemistry oncontrolled pore glass (CPG) supports in an automated DNA oligonucleotidesynthesizer (Generator™, Du Pont Co., Wilmington, Del., and Model 392,Applied Biosystems, Inc., Foster City, Calif.) (Beaucage and Caruthers,Tetrahedron Lett., 22 (20), 1859 (1981); Caruthers et al, GeneticEngineering, vol. 4, ed., (1982); Stelow and Hollaender, PlenumPublishing Corp., New York). The amino-modifying phosphoramidite reagentAminolink 2™ is obtained from Applied Biosystems, Inc., Foster City,Calif. Oligonucleotides are radio-labeled with [α³² P] cordycepin5'-triphosphate and scintillation fluid (Biofluor™) for scintillationcounting is obtained from NEN Products, Du Pont Co., Boston, Mass. andscintillation counting is accomplished using a Beckman Model LS3801scintillation counter (Beckman Instruments, Inc., Palo Alto, Calif.).Deoxynucleotydyl transferase is obtained from Promega, Inc., Madison,Wis. Poly-tergent SLF-18 is obtained from Olin Corp., Stamford, Conn.Kodak Xomat™AR 2 X-ray film for autoradiography is obtained from EastmanKodak Co., Rochester, N.Y. The reagents, SATA (N-succinimidylS-acetylthioacetate) and SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) are obtained from Pierce, Rockford, Ill. TheSATA and SMCC coupling chemistry is described by Tseng et al., incommonly owned U.S. Ser. No. 07/946247. Reporter antibody, used in thereporter conjugate (AffiniPure Goat anti-Rabbit IgG, H+L) is obtainedfrom Jackson ImmunoResearch Labs., Inc.; Product No.:111-005-045.Antibody reaction components are separated from the oligonucleotidecomponents by high-pressure liquid chromatography (HPLC) using a Zorbax250 Gel Exclusion column (9.4×250 mm, with 0.2M sodium phosphate bufferpH 7.0 and a column flow rate of 1 ml/min.) (MacMod Analytical Inc.Chadds Ford, Pa.) connected to a Waters 600 E System controller and aWaters 991 Photodiode Array detector (Millipore Corp., Milford, Mass.).Injections were made with a Waters 700 Satellite WISP--automatedinjection system. Test beads of glycidyl methacrylate, (oxirane acrylicbeads) functioning as the immobilized capture are purchased from Sigma(Product No. O-9754, Sigma Chemical Co., St. Louis, Mo.). Densitometersused in the following examples were either a Densigraph 100™ (GraphicTechnology Inc., Cherry Hill, N.J.) or the Model RD107R Quanta LogDensitometer (MacBeth Corp., Newbough, N.Y.). Basic polymerase chainreaction (PCR) protocols are described in Saiki, R. S. Scharf, F.Faloona, K. Mullis, G. Horn, H. A. Erlich, and N. Amheim, 1985. Science230:1350 and the amplification reaction is done using reagents obtainedin the Perkin Elmer-Cetus GeneAmp® kit (N801-0055), the PerkinElmer-Cetus 9600 GeneAmp® PCR System thermal-cycler (Perkin Elmer-Cetus,Norwalk, Conn.). PCR protocols used in the present invention weremodified to amplify short (<150 bases) single-stranded DNA targetsequences. In all of the examples provided below, the ADRS assay isdemonstrated using polymerase chain reaction (PCR) replicationtechniques, however it should be understood that any suitable method ofnucleic acid replication may be used including Ligase Chain reaction,and isothermal or autocatalytic methods.

Example 1 Amplified Analyte Detection In A Mouse IgG Assay Using DirectTarget Deposition Method

Oligonucleotide Preparation

While not intending to be limiting, and for illustration only, thesequences and primers used in replication could be synthesized with thefollowing sequences.

                            Target Sequence for Single Primer Amplification:                                -    1                                                                                    45                                        5' ATG CGT AGC AGC TTT ACC GCA GAG ATC ATG CCT ATG TAC CAT GCT 3'                                    3' TAC GCA TCG TCG AAA TGG CGT CTC TAG TAC GGA                               TAC ATG GTA CGA 5'                                       -    46                                   75                                 5' ATC CTA CCT GTA AGT AAA GCT GCT ACG CAT 3' SEQ ID NO:3                     3' TAG GAT GGA CAT TCA TTT CGA CGA TGC GTA 5' SEQ ID NO:4                      - Target/Primer Binding Sites for Single Primer                              Amplification:                                                                 -    1                                                        45                                    5' ATG CGT AGC AGC TTT ACC GCA GAG ATC ATG CCT                               ATG TAC CAT GCT 3'                                      3' TAC GCA TCG TCG AAA TGG CGT CTC TAG TAC GGA TAC ATG GTA CGA 5'                                    5' ATG CGT AGC AGC TTT AC  3' Primer 1 SEQ ID                                NO:5                                                     -                  3' CA TTT CGA CGA TGC GTA 5'  SEQ ID NO:5                    46                                   75                                    5' ATC CTA CCT GTA AGT AAA GCT GCT ACG CAT 3' SEQ ID NO:3                     3' TAG GAT GGA CAT TCA TTT CGA CGA TGC GTA 5' SEQ ID NO:4                      - Target Sequence for Double Primer Amplification:                            - 5' ATG CGT AGC AGC TTT ACC GCA GAG ATC ATG CCT ATG TAC CAT GCT 3'                                 3' TAC GCA TCG TCG AAA TGG CGT CTC TAG TAC GGA                               TAC ATG GTA CGA 5'                                       - 5' ATC CTA CCT GTA AGT CAT AGC TGT TTC CTG 3' SEQ ID NO:1                  3' TAG GAT GGA CAT TCA GTA TCG ACA AAG GAC 5' SEQ ID NO:2                      - Primer 1:                                                                   -      5' ATG CGT AGC AGC TTT AC 3' SEQ ID NO:5                               - Primer 2                                                                    -      3' CAG TAT CGA CAA AGG AC 5' SEQ ID NO:6                               - Target Sequence/Primer Binding Sites for Double Primer                     Amplification:                                                                 - 5' ATG CGT AGC AGC TTT ACC GCA GAG ATC ATG CCT ATG TAC CAT GCT 3'                                 3' TAC GCA TCG TCG AAA TGG CGT CTC TAG TAC GGA                               TAC ATG GTA CGA 5'                                      5' ATG CGT AGC AGC TTT AC  3'  Primer 1 SEQ ID NO:5                            -                  3' CA GTA TCG ACA AAG GAC 5' Primer 2 SEQ ID NO:6                                5' ATC CTA CCT GTA AGT CAT AGC TGT TTC CTG 3'                                SEQ ID NO:1                                             3' TAG GAT GGA CAT TCA GTA TCG ACA AAG GAC 5' SEQ ID NO:2               

Many alternative base sequences and chain lengths can also be employedwithin the guidelines discussed herein.

Nucleic Acid Replication Composition

The replication composition useful in amplifying the sequence target inpolymerase-type amplifications may comprise a solution containingreplication buffer (25 mM KCl, 20 mM TRIS hydrochloride [pH 8.13], 1.5mM MgCl₂, 0.05% Tween 20 and 0.1 mg/ml autoclaved gelatine [wt/vol]),200 mM each of the dNTPs, 1.0 uM of each primer, 2.5 U of Taq DNApolymerase made up in double-distilled water containing (0.1% wt/vol.)diethylpyrocarbonate. When nucleic acid replication is carried out usinglabeled deoxynucleotide triphosphates, the above replication compositionmay be modified so that one or more of the deoxynucleotide triphosphates(dNTPs) is replaced with labeled bases. Biotinylated dUTP (bio-dUTP)(Enzo Biochem, New York, N.Y.) and fluorescein labeled dCTP (F-dCTP)(Boehringer Mannheim Biochemicals, Indianapolis, Ind.) can be obtainedfrom Bethesda Research Laboratories, Inc., Maryland, and may besubstituted respectively for dTTP and dCTP in a 1:3 molar ratio oflabeled to unlabeled base. Generally, a 20 to 30% incorporation oflabeled bases is preferable in order to maintain efficient hybridizationV. T. Chan et al., Nucleic Acids Res., 13, 8083 (1985).

Nucleic Acid Amplification

Target and reference sequence replication may be performed using a DNAThermal Cycler and Gene Amp Kit using native Taq polymerase(Perkin-Elmer Cetus Corp., Norwalk, Conn.).

For amplification, 50 to 100 ul of the above replication compositioncould be added to ADRC test well containing the immobilized targetsequence. The replication composition is agitated to assure contact withthe support, and then overlaid with 75 ul of mineral oil (BDH Paraffinoil). The nucleic acid sequences are then denatured at 94° C. for 1 to 3min. A total of 25 to 10 temperature recycles could be performed underthe following conditions: denaturation at 94° C. for 0.5 to 1 min.,primer annealing at 37 to 60° C. for 0.5 to 1 min., DNA extension at 72°C. for 1 to 2 min.

Preparation of N-Hydroxysuccinimide-Activated Target Nucleic Acid

Preparation of a 5' activated spacer arm can be accomplished bysubstituting thymidine bases in the target sequence with C5 thymidineanalogs substituted with a C12 spacer linker arm terminating in anactive esters as described by J. Ruth et al., Fed. Proc., 44 (5), 1622(1985). The C5 amino-modified thymidine analog can be syntheticallyincorporated into the oligonucleotide sequence using conventionalphosphoramidite activation chemistry (Applied Biosystems, Foster City,Calif., Model 392 DNA synthesizer) and then derivatized with DSS(disuccinimidyl suberate) Pierce, Rockford, Ill., as described by Ruthet al., (1985) to form an active N-hyroxysuccinimide (NHS) ester.

Preparation of Antibody/Nucleic Acid Reporter Conjugate

A solution of the NHS oligonucleotide (0.5 umole equivalents) may becoupled with 0.25 umoles of goat anti-mouse IgG (Fab fragment specific)antibody (ICN) in a 1M NaHCO₃ buffer at pH 9.0. The reaction mixture isthen incubated for 2 hr at room temperature in the dark. The antibodyreporter conjugate is purified away from free nucleic acid and antibodyusing polyacrylamide (4 to 7%) gel electrophoresis under non-denaturingconditions (TBE) buffer). The conjugate is recovered by cutting out theconjugate band and placing it into a stoppered Econo-column (Bio-Rad)containing phosphate buffer saline solution (pH 7.4) as described by G.H. Keller and Manak, M. M., in DNA Probes, (1989), pp. 129-142, StocktonPress New York. The conjugate is then concentrated by centrifugationusing a prewashed Centricon 10 (Amicon) micro-concentrator.

Polystyrene beads (approximately 1-4μ microsphere beads, PolySciences,Inc., Warrington, Pa.) are coated with goat anti-mouse IgG (Fc fragmentspecific) antibody (Sigma Chemical, St. Louis, Mo.) in 0.1 carbonatebuffer pH 9.6 to prepare the solid phase antibody supports. Afterincubating overnight at 4° C. the antibody solution is removed bycentrifugation and aspiration. The beads are blocked with a solution of2% bovine serum albumin (BSA) dissolved in the above carbonate buffer.The beads are then washed free of excess reagents by rinsing three timeswith a 10 mM phosphate buffered (pH 7.4) saline solution with 0.05%Tween (PBST) and then resuspended in PBST. Dilutions of mouse IgG (mIgG)are made by dilution into PBST solution containing 1% BSA (BSA-PBST).Aliquots of mouse IgG solution are added to tubes containing themicrosphere beads, to yield a range of mouse IgG concentrations from 0,0.001, 0.01, 0.1, 1.0 to 10 ng/tube of mIgG. A solution (5 ul)containing 0.01 ug/ml of the antibody/target reporter conjugatedescribed previously is then added, and the assay mixture is incubatedfor 1 hour. Excess reagent is then removed by washing the microbeadswith three exchanges of PBST wash fluid.

Target replication is then achieved by adding 100 ul of the nucleic acidreplication composition to test microsphere beads. The test solutionsare mixed to assure contact with the support and then overlaid with 50ul of mineral oil (BDH Parrafin oil). The target sequence is thendenatured at 94° C. for 1 to 3 min. The reaction mixtures are thermallycycled using a DNA Thermal Cycler (Perkin-Elmer Cetus Corp., Norwalk,Conn.) according to manufacturer instructions.

Measurement of test response in target sequence-amplified wells could beachieved following equilibration with the above sequence amplificationcomposition modified to contain biotinylated dATP and fluoresceinlabeled dCTP as described above.

For detection of the amplified signal-generating nucleic acids, theamplification reaction fluid in each well is removed and transferred tomicrotiter plate wells coated with streptavidin as follows: PolystyreneEIA Microtiter Strips (NUNC) are filled with streptavidin (Sigma, St.Louis, Mo.) solution prepared in 0.1M carbonate buffer pH 9.6. Afterincubating overnight at room temperature (RT) the solution can beremoved and the strips blocked with a solution of 2% bovine serumalbumin (BSA) dissolved in the above carbonate buffer. The strips canthen be washed free of excess reagents by rinsing three times with PBST.The test fluids are then incubated in the avidin coated wells for 30min. and then washed free of unbound target. The wells are then filledwith 10 mM phosphate buffer (pH 7.4) saline (PBS) buffer, and thefluorescence in each well is measured.

Assay response in this example would result from binding of the antibodyreporter conjugate to the surface of the test wells. The target sequencecontained on the reporter conjugate is replicated using replicationcomposition containing biotinylated bases and bases labeled with afluorophore as previously described. The incorporation of these basesduring sequence replication results in the formation of nucleic acidscontaining both biotinylated and fluorescent bases. For detection, thenucleic acids would be isolated by capture onto an avidin solid-phasesupport, and could then be detected by measuring the signal nucleic acidfluorescence. Assay intensity would increase in proportion to theconcentration of mouse IgG analyte in the sample.

Example 2 Amplified Analyte Detection In A Mouse TgG Assay Using TheIndirect Target Deposition Method

Preparation of Tyramine Nucleic Acid Substrate

A solution of the NHS target oligonucleotide (0.5 umole equivalents) canbe coupled with 0.5 umoles tyramine (recrystallized from water, Aldrich,Milwaukee, Wis.) in 2.5 ml of dimethylformamide by addition of 1.0 ml of1M triethylammonium bicarbonate, pH 7.5 and then heated at 50° C. for 3hours. The solution can then be concentrated to dryness on a rotaryevaporator and purified by recrystallization from water or by HPLC usinga reverse-phase column in a Perkin-Elmer high performance liquidchromatograph.

Preparation of Streptavidin Nucleic Acid Conjugate

A solution of the NHS oligonucleotide (0.5 umole equivalents) could becoupled with 0.25 mmoles of streptavidin (Sigma) in a 1M NaHCO₃ bufferat pH 9.0. The reaction mixture can be incubated for 2 hr at roomtemperature in the dark. The conjugate can be purified away from freesequence and avidin using polyacrylamide gel eleclrophoresis undernon-denaturing conditions using a TBE buffer as described by M. S.Ureda, Methods in Enzymol., 146, 22 (1987) and M. S. Ureda et al.,Nucleic Acids Res., 16, 4937 (1988).

Polystyrene microsphere beads (approximately 1-4μ) are coated with goatanti-mouse IgG (Fc fragment specific) antibody in 0.1M carbonate bufferpH 9.6. After incubating overnight at room temperature the antibodysolution is removed by centrifugation and aspiration. The microbeads areblocked with a solution of 2% bovine serum albumin (BSA) dissolved inthe above carbonate buffer. The beads are then washed free of excessreagents by rinsing three times with PBST. Antigen dilutions of mouseIgG are prepared in PBST containing 1% BSA (BSA-PBST) as described inExample 1, and are then added to two sets of tubes containing the beads.The beads are incubated at 37° C. for 1 hour, followed by washing withPBST. Goat anti-mouse IgG-HRP (Boehringer Mannheim) is diluted, asrecommended by the manufacturer, and incubated for 1 hour at 37° C.Excess reagent is then removed by washing and aspirating three timeswith PBST.

A stock solution of biotin-tyramine conjugate (1 mg/ml) in dimethylsulfoxide is then prepared as described above. Just before use, thestock solution is diluted in 0.1M borate buffer pH 8.5 containing 0.01%H₂ O₂ to prepare a substrate solution containing 10 ug/mlbiotin-tyramine. The substrate solution is then added to both sets ofthe test beads and incubated for 15 minutes at room temperature.Unreacted substrate is then removed and the test beads washed with PBSTat 37° C.

For a comparison of detection sensitivity, streptavidin-alkalinephosphatase (Sigma Co., St. Louis, Mo.) is diluted as recommended by themanufacturer and added to one set of test beads as a reference control.For target nucleic acid replication, streptavidin-target sequenceconjugate (1 ug/ml) in PBST is added to the second set of beads. Bothsets of test beads are then incubated at room temperature for 30 minutesand then washed 3 times with PBST to remove unreacted conjugate.

Measurement of response in reference control wells is then achieved byaddition of p-nitrophenyl phosphate solution (1 mg/ml) in 10 mMdiethanolamine (pH 9.5), 0.5 mM MgCl₂ buffer to both sets of test beads.After 15 minutes at 37° C., color development is stopped by addition of50 ul of 0.1M EDTA and optical densities are read at 405 nm in amicrotiter plate reader (Molecular Devices Corp., California).

Measurement of test response in target nucleic acid amplified wells isachieved by first equilibrating the second set of beads with thereplication composition as described in Example 1 above. The testmixtures are agitated to assure contact with the bead supports and thenoverlaid with 75 ul of mineral oil (BDH Paraffin oil). The targetsequence is denatured at 95° C. for 1 to 3 min. The reaction mixturesare thermally recycled 30 times using a DNA Thermal Cycler according tomanufacturer instructions. For detection of the amplified target nucleicacids, 1 ul of a stock solution of 100 ml of ethidum bromide (0.5 mg/ml)in a Tris Acetate EDTA buffer (pH 8.1) containing 40 mM Tris base, 2 mMacetic acid, 0.2 mM EDTA is added to the reaction supernatant. Thesolution is then excited with shortwave light at 254 nm and thefluorescence is detected.

Assay response in this example would result from the ADRC-HRP catalyzeddeposition of biotin/tyramine reporter on the test bead surface followedby subsequent binding of the streptavidin signal-generating sequencetarget. The target is then amplified using nucleic acid replicationcomposition as described in Example 1 and the resulting nucleic acidproducts are detected by dye intercalation. Assay intensity wouldincrease in proportion to the concentration of mouse IgG in samples, andcould be detected at mIgG concentration below that which is detectableusing an non-amplified ADRC-AP reporter.

Example 3 Amplified Analyte Detection In A Mouse IgG Assay UsingCatalyzed Direct Target Deposition Method

Polystyrene microspheres (1-4μ) are coated with goat anti-mouse IgG (Fcfragment specific) antibody in 0.1M carbonate buffer pH 9.6. Afterincubating overnight at room temperature (RT) the antibody solution isremoved and the strips blocked with a solution of 2% bovine serumalbumin (BSA) dissolved in the above carbonate buffer. The beads arethen washed free of excess reagents by rinsing three times with a 10 mMphosphate buffered (pH 7.4) saline solution containing 0.05% Tween 20(PBST). Antigen dilutions of mouse IgG (mIgG) dissolved in a solutionPBST containing 1% BSA (BSA-PBST) are then added to the beads asdescribed in Example 1. The beads are then incubated at 37° C. for 1hour followed by washing 3 times with PBST, and treated with goatanti-mouse IgG-HRP as described in Example 2.

For catalyzed reporter deposition, a stock solution of tyramine-genetarget substrate (1 mg/ml) in dimethyl sulfoxide is prepared. Justbefore use, the stock solution is diluted in 0.1M borate buffer pH 8.5containing 0.01% H₂ O₂ to prepare a substrate solution containingtyramine-sequence target substrate (10 ug/ml). The substrate solutionsis then added to the test beads and incubated for 30 minutes at roomtemperature. The reaction mixture is then removed and the test wellswashed with PBST at 37° C.

Measurement of test response in sequence amplified wells is achievedfollowing equilibration of each set of test beads with the abovesequence replication composition modified to contain biotinylated andfluorescein labeled nucleotides as described in Example 1 above. Thetest solutions are mixed to assure contact with the support and thenoverlaid with 75 ul of mineral oil (BDH Paraffin oil). The targetsequence is then denatured at 95° C. for 1 to 3 minutes, and then thereaction mixtures are thermally recycled 30 times using a DNA ThermalCycler according to the manufacturer instructions.

For detection of the amplified signal-generating nucleic acids, thereplication reaction fluid in each set of test beads well is removed andtransferred to microtiter plate wells coated with streptavidin asfollows: Polystyrene EIA microtiter plate wells are filled withstreptavidin (Sigma) solution prepared in 0.1M carbonate buffer pH 9.6.After incubating overnight at room temperature (RT) the streptavidinsolution is removed from each well and microtiter wells blocked with asolution of 2% bovine serum albumin (BSA) dissolved in the abovecarbonate buffer. The wells are then washed free of excess reagents byrinsing three times with a 10 mM phosphate buffered (pH 7.4) salinesolution containing 0.05% Tween 20 (PBST). Once prepared the above testfluids are then transferred and then incubated in the avidin-coatedwells for 30 minutes and then washed free of unbound target replicationreagents. The wells are then filled with PBS buffer, and thefluorescence in each well is measured.

Assay response in this example would result from the ADRC-HRP catalyzeddeposition of tyramine-sequence target reporter on the test beadsurface. The immobilized target nucleic acid is then amplified usingsequence replication composition containing biotinylated bases and baseslabeled with a fluorophore. The incorporation of these bases duringsequence replication would result in nucleic acids containing bothbiotin and fluorescent-labeled bases. For detection, the nucleic acidsare isolated by capturing on an avidin solid-phase support and thendetected by measuring the nucleic acid fluorescence. Fluorescentintensity increases with the concentration of mouse IgG analyte in thesample.

Example 4 Detection and Quantitation of Mouse IgG Using A ReferenceInternal Control Assay

Preparation of biotin, isotope and fluorescence-labeled bases

The target or reference sequences, and primer sequences can be designedto enable incorporation of biotin, isotope or (and) fluorescence labeledbases or primers. In this way, the sequence replication process canproduce nucleic acid strands providing means of both capture anddetection. For example, either the target nucleic acid or a referencesequence could be prepared with the following bases:

                          Reference Nucleic Acid/Primer Binding Sites:                                    -    1                                                                                  45                                            5' ATG CGT AGC AGC TTT ACC GCA GAG ATC ATG CCT ATG TAC CAT GCT 3'                                  3' TAC GCA TCG TCG AAA TGG CGT CTC TAG TAC GGA TAC                           ATG GTA CGA 5'                                            5' ATG CGT AGC AGC TTT AC 3' Primer 1 SEQ ID NO:5                              -                  3' AT CAT CTT TGT CGA CTG 5' Primer 5 SEQ ID NO:13                                46                                   75                                    5' ATC CTA CCT GTA ATA GTA GAA ACA GCT GAC 3' SEQ                            ID NO:7                                                   3' TAG GAT GGA CAT TAT CAT CTT TGT CGA CTG 5' SEQ ID NO:8                      - Primer 1 (capture):                                                         -      5' biotin-ATG CGT AGC AGC TTT AC 3' SEQ ID NO:5                        - Primer 5 (reporter):                                                        -      3' AT CAT CTT TGT CGA CTG-Fluorophore (Flu) 5' SEQ ID NO:13                                 - Target Sequence/Primer Binding Sites:                  -     5' ATG CGT AGC AGC TTT ACC GCA GAG ATC ATG CCT ATG TAC CAT GCT                             3'                                                            3' TAC GCA TCG TCG AAA TGG CGT CTC TAG TAC GGA TAC ATG GTA CGA 5'                              biotin-ATG CGT AGC AGC TTT AC 3' SEQ ID NO:5                                   -                      3' AT CAT CTT TGT CGA                                CTG-Flu 5' SEQ ID NO:13                                       5' ATC CTA CCT GTA ATA GTA GAA ACA GCT GAC 3' SEQ ID NO:7                     3' TAG GAT GGA CAT TAT CAT CTT TGT CGA CTG 5' SEQ ID NO:8           

Alternatively, sequences could be designed so that the positions offluorescent-labeled bases are in appropriate spatial alignment forefficient energy transfer between fluorophores.

Polystyrene microsphere beads (1-4μ diameter) are coated with goatanti-mouse IgG (Fc fragment specific) antibody (ICN) in 0.1 carbonatebuffer pH 9.6. After incubating overnight at room temperature the IgGsolution is removed and the beads blocked with a solution of 2% bovineserum albumin (BSA) dissolved in the above carbonate buffer. The beadsare then washed free of excess reagents by rinsing three times with 10mM PBST. For calibration, a standard curve is established by determiningthe assay response from a series of samples containing knownconcentrations of mouse IgG (mIgG). To prepare the standard solutions,dilutions of mIgG are made by dissolving mIgG in PBST solutioncontaining 1% BSA (BSA-PBST). Each standard is added to the samequantity of test beads. In this way, samples containing a range of mouseIgG concentrations from 0, 0.001, 0.01, 0.1, 1.0 to 10 ng/tube of mIgGmay be prepared. A test sample containing an unknown concentration ofmIgG is also added to a separate set of beads.

The tubes containing both the test and standard beads are then incubatedwith 10 ul of a solution (0.1 ug/ml) of the above anti-mIgGantibody/target reporter conjugate. Excess reagents are then removed bywashing with PBST.

Amplification of the target nucleic acid on both the antibody targetreporter conjugate and a reference sequence is achieved by adding totest wells 100 ul of a replication composition which also containsreplication control at appropriate sequence and primer concentrations.The test solutions are mixed to assure contact with the support and thenoverlaid with 50 ul of mineral oil (BDH Parrafin oil). The target andreference sequences can then be denatured at 94° C. for 1 to 3 minutes,and the reaction mixtures are thermally recycled 30 times using a DNAThermal Cycler according to manufacturer instructions as described inExample 1 above.

In this example, the capture primer #1 for both the target and referencenucleic acid are identical, and prepared so as to contain biotinylateddUTP at the 5' end of the primer strand. The reporter primers (#2) forthe two different targets will comprise different sequences, onespecific for the target nucleic acid, and one specific for the referencenucleic acid. Each primer sequence is complementary for the antisensestand and its respective target. Both the reporter primer and referenceprimer can be amino-modified at their 5' end with amino-modifyingphosphoramidite reagent during their synthesis on an automated DNAsynthesizer. The amino-modified reporter primer can be reacted with afluorescein NHS ester, labeling the 5' end of the primer withfluorescein. The amino-modified reference primer can be labeled with arhodamine fluorophore NHS ester using the same chemistry.

For detection of the amplified nucleic acids, the amplification reactionfluid in each test well is removed and transferred to microtiter platewells previously coated with streptavidin as follows: Polystyrene EIAMicrotiter Strips (NUNC) are filled with streptavidin (Sigma, St. Louis,Mo.) solution prepared in 0.1M carbonate buffer pH 9.6. After incubatingovernight at room temperature the streptavidin solutions are removed andstrips blocked with a solution of 2% bovine serum albumin (BSA)dissolved in the above carbonate buffer. The strips can then be washedfree of excess reagents by rinsing three times with a 10 mM phosphatebuffered (pH 7.4) saline solution containing 0.05% Tween 20 (PBST). Thetest fluids resulting from the above nucleic acid replications are thenincubated in the streptavidin coated wells for 60 minutes and thenwashed free of unbound signal nucleic acid products and reagents. Therhodamine and fluorescein fluorescence in each well is measured for boththe standards and the test sample, and the ratio of rhodamine tofluorescein fluorescence in the standard samples and test samples iscomputed. The fluorescein response in the test sample would then becorrected based upon the average rhodamine to fluorescein responsedetermined in the standard samples. The corrected fluorescein responsewould then be used to determine the mIgG concentration by interpolationof fluorescein response measured in the mIgG standard wells. Fluoresceinintensity in test samples would increase in proportion to theconcentration of mouse IgG analyte in the sample.

Example 5 Preparation of a 75 base Oligonucleotide Reporter-AntibodyConjugate Using Heterobifunctional Crosslinking Chemistry, for use inthe Direct Target Deposition Method

Oligonucleotide Synthesis or Preparations

The 75 base oligonucleotide used as a nucleic acid reporter (target) wasamino-modified at the 5' end, that is, a primary amine group wasintroduced at the 5' end of the oligonucleotide target. The primaryamine group was later used in the NHS-heterobifunctional chemistry tocouple the DNA target to the test antibody(Ab). Amino-modification wasaccomplished using the CE phosphoramidite chemistry during the synthesisof the target on the automated DNA synthesizer (Smith, L. M., S. Fung,M. W. Hunkapiller and L. E. Hood (1985) Nucleic Acids Res. 11:2399-2412;Sproat, B. S., B. Beijer and P. Rider (1987) Nucleic Acids Res.15:6181-6196). Aminolink 2™ (Applied Biosystems) an amino-modifyingphosphoramidite reagent was incorporated during the last phosphoramiditecoupling cycle of the oligonucleotide synthesis.

The following target primer sequences were designed and synthesized tobe used as the target sequence in the conjugate reporter.

                       Target Sequence (75mer) for Double Primer Conjugate                            Reporter system:                                             - 5'X-GGC AGG AAG ACA AAC ACT GGC TGG TCT GTG GTG CTG TGC TTG TTC CCC                         TGT                                                            ..CCT AGT ATT GTT TTC TGG GTT GGT 3' SEQ ID NO:9                             - (X = Aminolink 2™ amino-modifer)                                         - Primer 3 (3' primer for the 75mer) (17mer) sequence:                        - 5'ACC AAC CCA GAA AAC AA 3' SEQ ID NO:10                                    - The primer binding site for Primer 3 (3' primer for the                    75mer) is illustrated below:                                                   - 5'X-GGC AGG AAG ACA AAC ACT GGC TGG TCT GTG GTG CTG TGC TTG TTC CCC                         TGT                                                            ..CCT AGT ATT GTT TTC TGG GTT GGT 3' SEQ ID NO:9                                       3'AA CAA AAG ACC CAA CCA 5' SEQ ID NO:10                     

The underlined sequence is the complementary sequence of the Primer 3 orthe "3' primer binding site".

The double stranded 75mer reporter produced from primer extension(replication) is illustrated below:

    5'X-GGC AGG AAG ACA AAC ACT GGC TGG TCT GTG GTG CTG TGC TTG TTC CCC TGT        3'  CCG TCC TTC TGT TTG TGA CCG ACC AGA CAC CAC GAC ACG AAC AAG GGG ACA        -   ..CCT AGT ATT GTT TTC TGG GTT GGT 3' SEQ ID NO:9                           ..GGA TCA TAA CAA AAG ACC CAA CCA 5' SEQ ID NO:11                            - Primer 4 (5' primer of the 75mer) (16mer) primer binding                   site is illustrated below:                                                     - 5'X-GGC AGG AAG ACA AAC ACT GGC TGG TCT GTG GTG CTG TGC TTG TTC CCC       TGT                                                                             3'  CCG TCC TTC TGT TTG TGA CCG ACC AGA CAC CAC GAC ACG AAC AAG GGG ACA      5'  GGC AGG AAG ACA AAC A 3' SEQ ID NO:12                                      - ..CCT AGT ATT GTT TTC TGG GTT GGT 3' SEQ ID NO:9                           ..GGA TCA TAA CAA AAG ACC CAA CCA 5' SEQ ID NO:11                        

The underlined sequence is the complementary sequence of Primer 4 or the"5' primer binding site".

The crude target and primer oligonucleotides were analyzed for fulllength products and failure sequences by 8% polyacrylamide/8.3M urea gel(denaturing) electrophoresis (Sanger, F and A. R. Coulson. 1978. FEBSLett. 87:107) and standard autoradiography. The oligonucleotides wereradio-labeled at the 3' end with [α³² P] cordycepin 5'-triphosphate(5000 Ci/mmol) using terminal deoxynucleotydyl transferase (TdT). Thiswas accomplished by adding 100 ng of the oligonucleotide to 10 ulreaction solution containing 100 mM cacodylate, pH 6.8, 1 mM CoCl₂, 0.1mM DTT, 100 μg/ml BSA and 10 units of TdT. The reaction was incubated at37° C. for 30 min. An aliquot, 2 ul, of the labeled oligonucleotides wasadded to 6 ul of a loading solution (90% formamide, 0.05% bromophenolblue and 0.05% xylene cyanol FF), mixed, heated (90° C. for 1 min.) andloaded on an 8% polyacrylamide/8.3M urea gel (40×30×0.04 cm) in 1× TBEbuffer (8.9 mM Tris-borate, pH 8.2, 2 mM EDTA). The electrophoresis wascarried out by applying 55 Watts (or 1.35 W/cm) to the gel for 2.5 hrsor until the bromophenol blue ran to the bottom of the gel. The gel wastransferred onto a piece of Whatman 3 MM paper (Whatman International,Ltd., Maidstone, England) and dried on a Model 583 gel dryer (Bio-RadLaboratories, Richmond, Calif.) at 80° C. for 1 hr. The dried gel andX-ray film (Kodak Xomat™AR 2) were placed in an X-ray cassettecontaining an intensifying screen (Du Pont Cronex® Lighting Plus™, DuPont Co., Wilmington, Del.), and the film was exposed for an appropriateamount of time to obtain an autoradiographic image.

Synthesis of the Reporter Conjugate

The rationale or the strategy used for synthesizing the reporterconjugate is the same as outlined in the specification. The protocolinvolves first coupling a sulfhydryl group to the 75-base amino-modifiedoligonucleotide using the SATA reagent followed by the addition of amaleimide group to the goat antibody using the SMCC reagent, and finallythe linking of the 75-base, SATA-modified oligonucleotide to themaleimide-modified goat antibody.

Coupling a Sulfhydryl Group to the 75-base, Amino-modifiedOligonucleotide

The 75-base amino-modified oligonucleotide, 1.4 mg (60 nmoles), wasadded to a 667 uL reaction mixture containing 100 mM sodium bicarbonatebuffer, pH 9.0, 13.3 mg SATA (N-succinimidyl S-acetylthioacetate) 50%dimethyl formamide(DMF). The SATA reagent was prepared by dissolving 20mg in 500 uL of DMF. The reaction mixture was allowed to proceed for 30min. at 25° C., then immediately applied to a Sephadex® G-25 (PharmaciaLKB, Uppsala, Sweden) column , 1×20 cm and eluted at room temperaturewith 100 mM sodium phosphate buffer pH 6.5 at a flow rate of 1 ml/min.Fractions were monitored by absorbance at 280 nm (Pharmacia LKB #2138Unvicord S Monitor) and collected on a Pharmacia Model Frac 100 fractioncollector. The first peak fractions (1.0 ml), containing theSATA-modified oligonucleotides were pooled and concentrated to ˜1.0 mlusing an Amicon Centricon™ 3 concentrator (Amicon, W. R. Grace & Co.,Danvers, Mass.). The Centricon™ 3s were placed in a SM24 rotor (Du PontSorvall, Newtown, Conn.) and spun in a Du Pont Sorval® RC5B RefrigeratedSuperspeed centrifuge, @ 7500 rpm (7000×g) for 45 min. at 20° C. Thesamples were pooled, placed in another set of Centricon™ 3s and spunagain for 45 min. using the same centrifuge protocol. The SATA-modifiedoligonucleotide concentrate (˜1.0 ml) was recovered using the protocolrecommended by the manufacturer (Amicon), and was saved at 20° C. in thedark until needed for the final DNA-Ab coupling protocol.

Coupling Maleimide Groups to the Goat Antibody

The reporter antibody, used in the reporter conjugate (AffiniPure Goatanti-Rabbit IgG, H+L, 1.5 mg/ml), 3 mg, was added to a 2.7 ml reactionmixture containing 100 mM sodium phosphate buffer, pH 7.0, 2 mg SMCC,1.5% dimethyl formamide(DMF). The SMCC was prepared by dissolving 5 mgin 84 uL of DMF (60 mg/ml). This reaction was started 75 min. after the75 base amino-modified oligonucleotide was reacted with the SATAreagent. The reaction mixture was allowed to proceed for 30 min. at 25°C., then immediately applied to a Sephadex® G-25 column, 1×20 cm andeluted at room temperature with 100 mM sodium phosphate buffer pH 6.5 ata flow rate of 1 ml/min. Fractions were monitored by absorbance at 280nm and collected on a Pharmacia Model Frac 100 fraction collector. Thefirst peak fractions (1.0 ml), containing the SMCC-modified(maleimide-modified) goat antibody were pooled (4 to 6 ml) into onetube. The reaction product was ready for coupling to the SATA-modifiedoligonucleotides.

Coupling the SATA-modified Oligonucleotides to the GoatMaleimide-modified Antibodies

The pooled maleimide-modified goat antibody fractions (5 ml) were addedto a 15 ml Falcon® 2059 tube (Becton and Dickinson and Co., LincolnPark, N.J.). The concentrated 75 base SATA-modified oligonucleotide(˜1.0 ml) was added to the same reaction tube and mixed well with themaleimide-modified goat antibody. The coupling reaction was initiated byadding 75 ul of 1M hydroxylamine (HA)(Pierce, Rockford, Ill.), pH 7.0,50 mM EDTA and mixing well. The reaction was transferred to an AmiconModel 3' MiniCell (6 ml stirred cell) concentrator fitted with a YM5filter (Amicon). The MiniCell was connected to a helium source, adjustedto 60 psi, and placed on a magnetic stirrer. The reaction was allowed toproceed while the reactions major components (modified-Abs,modified-oligonucleotides and newly formed DNA/Ab conjugate) were beingconcentrated at room temperature covered with aluminum foil. Thereaction volume was reduced to approximately 1.0 ml (60 min.). Thereaction was removed from the MiniCell and transferred to a Wheaton224812, amber 4.0 ml vial (Wheaton, Millville, N.J.) and incubated atroom temperature on a Lab Quake™ (Labindustries, Inc., Berkeley,Calif.), rotating until the total reaction time reached 2 hrs. Thereaction was terminated by the addition of 10 ul of 10 mMN-ethylmaleimide in DMF.

Isolation of the Oligonucleotide-Antibody Conjugates from the ModifiedOligonucleotide Component

The 75-base-oligonucleotide-goat-antibody conjugate (the reporterconjugate or the oligonucleotide-antibody conjugate) and the antibodyreaction components were separated from the oligonucleotide componentsby high-pressure liquid chromatography(HPLC). This was achieved using aZorbax 250 Gel Exclusion column (9.4×250 mm, with 0.2M sodium phosphatebuffer pH 7.0 and a column flow rate of 1 ml/min.) connected to a Waters600 E System controller and a Waters 991 Photodiode Array detector.Injections (200 ul) were made with a Waters 700 SatelliteWISP--automated injection system. The first peak fractions (0.5 ml)resulted in a mixture of the oligonucleotide-antibody conjugate and themaleimide modified-antibody reaction component.

The fractions were further analyzed for the oligonucleotide-antibodyconjugate by gel electro-phoresis and standard autoradiography. Theconjugate-linked oligonucleotide was radio-labeled at the 3' end with[α³² P] cordycepin 5'-triphosphate (5000 Ci/mmol) using TdT. This wasaccomplished by adding 2 ul of the HPLC-isolated fraction to a 10 ulreaction solution containing 100 mM cacodylate, pH 6.8, 1 mM CoCl₂, 0.1mM DTT, 100 μg/ml BSA, 10 units of TdT and 1 μCi of [α³² P] cordycepin5'-triphosphate. The reaction was incubated at 37° C. for 30 min. Thesamples were then analyzed on a standard sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE; Laemmli, U.K. 1970Nature 227:680-685) and standard DNA denaturing (urea) polyacrylamidegels (Sanger, F and A. R. Coulson,1978, FEBS Lett. 87:107).

An aliquot, 4 ul, of the labeled products was added to 12 ul of proteingel loading buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.01%bromophenol blue). The samples were loaded on a SDS-PAGE protein gel (8%polyacrylamide separation gel (15×15×0.075 cm), 375 mM Tris, pH 8.8,0.5% SDS; 4% polyacrylamide stacking gel, 0.12M Tris, pH 6.8, 1% SDS; inLaemmli running buffer, 0.25 mM Tris.HCl,192 mM glycine, 0.1% SDS, pH8.3). The electrophoresis was started at 100 V (6.7 V/cm.) and wasincreased to 225 V (15 V/cm.) after the sample moved through thestacking gel. The electrophoresis was continued until the dye front hadmigrated approximately 13-14 cm.

A second aliquot of 2 ul was added to 6 ul of a loading solution (90%formamide, 0.05% bromophenol blue and 0.05% xylene cyanol FF), mixed,and loaded on a 8% polyacrylamide/8.3M urea gel (40×30×0.04 cm) in 1×TBE buffer (8.9 mM Tris-borate, pH 8.2, 2 mM EDTA). The gelelectrophoresis was carried out by applying 55 Watts (or 1.35 W/cm) tothe gel until the bromophenol blue ran to the bottom of the gel.

For autoradiography, both the SDS-PAGE protein gel and the DNAdenaturing PAGE gel were transferred onto a piece of Whatman 3MM paperand dried on a Bio-Rad Model 583 gel dryer at 80° C. for 1 hr. The driedgel and X-ray film (Kodak Xomat™AR 2) were placed in an X-ray cassettecontaining an intensifying screen (Du-Pont Cronex® Lighting Plus™), andthe film was exposed for an appropriate amount of time to obtain anautoradiographic image.

The spectrophotometric scans of the HPLC fraction and data obtained fromthe autoradiography procedures described above were used to determinewhich HPLC fractions contained the oligonucleotide-antibody conjugatefree of the 75-base SATA-modified oligonucleotide precursor. The peakfractions were pooled and stored at 4° C.

Example 6 Amplification of a Rabbit IgG Assay using the Direct TargetDeposition Method

Preparation of Immobilized Capture Reagent for Immunoassays

The immobilized capture reagent (test beads) were prepared by adding 100mg of glycidyl methacrylate beads (oxirane acrylic beads 30μ), to a 500ul solution of the capture antibody, goat anti-Rabbit IgG antibody (0.5mg) (AffiniPure Goat anti-Rabbit IgG, H+L, 1.5 mg/ml) in PBS buffer. Thetest beads were incubated with rotation for 20 hrs. at 4° C. The excessgoat antibody was removed by centrifugation and aspiration. The testbeads were then pretreated with bovine serum albumin (BSA) to pacify theunreacted epoxide groups. The test beads were incubated with a 1 mg/mlsolution of BSA in PBS (10 mM phosphate buffered (pH 7.4) salinesolution) buffer for 2 hr at room temperature. The test beads werewashed with water to remove the BSA, then washed 4× with PBS buffer, andfinally resuspended in 1.0 ml of PBS buffer (0.1 mg/gL) containing 0.02%azide.

The Immobilized Capture Reagent's Immuno-Reactivity and ImmunoassayProcedure

The test beads' immuno-reactivity is assayed in two replicates by adding0.5 mg of the test beads to 250 ul Tris sample buffer (TSB) (50 mMTris.HCl, 75 mM sodium chloride, 0.1% poly-tergent SLF-18, 0.1% BSA and0.02% azide) in 500 ul "eppendorf" tubes (Eppendorf®, BrinkmannInstruments Co., Westbury, N.Y.). To assay tubes receiving the testantigen, Rabbit IgG (Purified Rabbit IgG), 20 ul of the stock solution(100 μg/ml) was added and the solutions were incubated at roomtemperature for 30 min. To the control assay tubes (test beads with noadded antigen), 20 ul of TSB buffer was added and the solutions wereincubated at room temperature for 30 min. The test beads were pellettedby centrifugation and the supernatants were removed by aspiration. Eachtest was washed 3× with TSB buffer. Each test was then incubated at roomtemperature with 50 ul of the goat anti-R-IgG- alkaline phosphataseconjugate (Sigma product No., A-8025) stock solution in a final reactionvolume of 250 ul for 1 hr. The conjugate reagent was removed bycentrifugation and aspiration and, then each test was washed 4× with TSBbuffer. The BCIP (bromochloroindoyl phosphate: Sigma product No.710-3)reagent (20 ul) was then added to each tube and incubated at roomtemperature for 30 min. The color (blue-green) developed in the test wasread to determine the degree of immuno-activity.

Determination of the Reporter Conjugate's Immuno-Activity and theImmunoassay's Signal to Noise Response

The oligonucleotide-antibody conjugate (75 base target oligonucleotideconjugated to the goat anti-R-IgG) was radio-labeled at the 3' end with[α³² P] cordycepin 5'-triphosphate (5000 Ci/mmol) using TdT. This wasaccomplished by adding 10 ul of the pooled HPLC peak fractions of theoligonucleotide-antibody conjugate to 20 ul reaction solution containing100 mM cacodylate, pH 6.8, 1 mM CoCl₂, 0.1 mM DTT, 100 μg/ml BSA, 30units of TdT and 20 μCi of [α³² P] cordycepin 5'-triphosphate. Thereaction was incubated at 37° C. for 30 min. Ten microliters of thelabeled oligonucleotide-antibody conjugate was added to 50 ul of "cold"conjugate. Serial dilutions from 5 ul down to 0.005 ul of theoligonucleotide-antibody conjugate were assayed for immuno-reactivity.For each dilution to be tested, two test replicates were set up byadding 0.5 mg of the test beads to 250 ul TSB buffer in 500 ul"eppendorf" tubes. To assay tubes receiving the test antigen, Rabbit IgG(Purified Rabbit IgG: Sigma, N I-5006), 20 ul of the stock solution (100μg/ml were added and the solutions were incubated at room temperaturefor 30 min. Two control assay tests (test beads with no added antigen)were made for each conjugate dilution. To each control test, 20 ul ofTSB buffer were added and the solutions were incubated at roomtemperature for 30 min. The test beads were pelletted by centrifugationand the supernatants were removed by aspiration. Each test was washed 3×with TSB buffer. Next, each test was incubated at room temperature for 1hr in 250 ul TSB buffer and the appropriate dilution of the [³² P]labeled oligonucleotide-antibody conjugate. The non-reacted, labeledreporter conjugate reagent was removed by centrifugation and aspiration,and then each test was washed 4× with TSB buffer. Each tube of assaytest beads or assay control beads was resuspended in 10 ul of TSB bufferand transferred to a scintillation vial containing 10 ml ofscintillation fluid (Biofluor™) The amount of radio-label in each testwas counted in a Beckman Model LS3801 scintillation counter (Beckman).The amount of signal and non-specific label for each dilution wasgraphically plotted. These data were used to determine a dilution of the75 base oligonucleotide-goat Ab conjugate at which the non-specificreactivity was indistinguishable from background. This dilution and theimmediate dilutions below this dilution of the reporter conjugate wereused in immunoassays followed.

Dose-Response Immunoassay using the Reporter Conjugate

Aliquots made from serial dilutions of the test analyte stock solution,rabbit IgG (100 μg/ml) (Sigma, N I-5006), 1 ug, 1 ng and 1 pg, wereadded to 500 ul tubes containing 0.5 mg of test beads in 250 ul TSB.There was also a control assay tube containing 250 ul of TSB buffer, 0.5mg test beads and no rabbit IgG. The assay solutions were incubated atroom temperature (antigen capture step) for 30 min. The test beads werepelletted by centrifugation and the supernatants were removed byaspiration. Each test was washed 3× with TSB buffer, and then incubatedat room temperature with 250 ul equivalent dilution of the TSBcontaining 0.02 ul of the oligonucleotide-antibody conjugate for 30 min.The supernatants were removed by centrifugation and aspiration. The testbeads were washed 4× with TSB and then 1× with water. The beads wereresuspended in 50 ul of water.

Target Sequence (Oligonucleotide) Amplification Procedure

Amplification of the 75-base-oligonucleotide target sequence conjugatedto the reporter antibody (goat anti-rabbit IgG, Jackson ImmunoResearchLabs.) was performed using the polymerase chain reaction (PCR). Theprotocol required two separate additions of primers. The first additionwas made before the initial cycle; the 3' primer was added at a 10×excess over the 5' primer. A second addition of primers was made afterthe fifteenth replication cycle with both primers added at the sameconcentration. The amplification reaction was done using reagents citedabove and the following conditions. For each test sample, a finalreaction volume of 50 ul (containing 10 mM Tris*HCl, pH 8.3, 50 mM KCl,1.5 mM MgCl₂, 0.001% gelatin, 100 μM dATP, 100 μM dCTP, 100 μM dGTP, 100μM dTTP and 0.25 units Taq DNA polymerase) was prepared. For the initialreaction, 2.5 ul of the 3' primer (400 nM stock sol.) and 2.5 ul of the5' primer (40 nM stock sol.) were added to a MicroAmp™ reaction tube(Perkin Elmer-Cetus) containing 25 ul of distilled water. Fivemicroliters of the test beads resuspended in water were added to theprimers and the tube was placed at 95° C. for five minutes. The lambdaprimers and DNA, the "kit" control, were also run as an additionalsample for the PCR reagent control (using the manufacturer's recommendedreactant concentrations for primers and lambda DNA). A master reactionmix was prepared using the Perkin Elmer-Cetus kit reagents and Taq DNApolymerase (Amplitaq®:Perkin Elmer-Cetus), ([n+1]×15 ul, where n equalsthe number of test samples) and was heated to 72° C. The master reactionmix was aliquoted (15 ul) to each sample tube, mixed and transferred tothe thermal-cycler block which was paused on hold at 72° C. After allsamples were added to the thermal-cycler, they were subjected to 15cycles of 90° C. for 15 sec. (denaturing conditions) and then at 42° C.for 10 sec. (primer annealing conditions). Since the target sequence wasshort (75 bases), an additional stage for polymerization was notrequired. Polymerization was accomplished during the ramp up to 90° C.for denaturation (<1° C./sec). After 20 cycles, the reaction was held at72° C. for the addition of 2.5 ul of the 3' primer (400 nm stock sol.)and 2.5 ul of the 5' primer (400 nM stock sol.). The reactions weresubjected to an additional 15 cycles using the same cycling programdescribed above. The reactions were then brought to 65° C. for 45 sec.and then to 4° C., holding for further analysis.

Analysis of Amplification Products

Amplification products were initially analyzed by submarine gelelectrophoresis. After the amplification of the 75 base target (thereporter oligonucleotide conjugated to the goat Ab), 15 ul of theamplified sample was mixed with 3 ul of agarose gel loading buffer (30%glycerol and 0.25% bromophenol blue) and analyzed on a 3% agarose gel(8.5×6.0×˜0.5 cm:25 ml agarose sol.) containing 0.1 μg/ml ethidiumbromide and 0.5× TBE buffer. The gel electrophoresis was carried out byapplying 50 V (or 5.9 V/cm) to the gel for 40 min. The results, ethidiumbromide-stained DNA bands, were visualized with a UV transilluminator(310 nm wavelength, Model TM-20, UVP, Inc., San Gabriel, Calif.) andrecorded on Polaroid type 57 black and white film (Polaroid Corp.,Cambridge, Mass.). The bands appeared on the film as white or light graybands on the dark gray to black background (the gel). Further analysisof amplification products was made by measuring the reflection densityof the ethidium bromide stained DNA bands on the Polaroid Type 57 filmusing a densitometer as cited above. The reflection densitometers werefirst standardized to standard density plaques that gave specificdensity reflections in density units. Next the gel background wasmeasured; then the ethidium bromide-stained DNA bands representing eachof the amplification product were measured. In this way, theamplification response of each assay was measured, reflecting the amountof antigen present in the sample when compared to a standard curve (adose-response curve).

Referring to FIG. 7, lanes 3 through 6 show the amplification responseof the 75-base reporter sequence using 0.02 ul of the conjugate inresponse to different amounts of antigen present in the immunoassay. Thesamples tested in Lanes 3, 4, and 5 contain 1 ug, 1 ng and 1 pg,respectively. Lane 6 is a control where no analyte was added to test forthe amount of non-specific binding (n.s.b.). Lane 1 is Hae III digestedΦ X174 molecular weight markers, and lane 2 is λ control primer dimer.

In Table 1 below, the results represent the dose-response of theimmunoassay of FIG. 7. Quantitation of the 75-base product was done bymeasuring the Polaroid 57 film for percent reflectance using the ModelRD107R Quanta Log Densitometer. The data expressed as the relativeintensity of the ethidium bromide stain (percent reflectance) indicatingthe assay response as a function of the amount of analyte (Rabbit IgG)present.

                  TABLE 1                                                         ______________________________________                                        Relative Band Density of Amplification Products                                Produced in Response to Increasing R-IgG Analyte                              Concentrations, in the Direct Target Method                                                 Amt. of Analyte                                                                           Band Density in                                      Lane (R IgG) Percent Reflectance                                            ______________________________________                                        6          0           9.5                                                      5 1.0 (1 pg) 10                                                               4 1.0 (1 ng) 18                                                               3 1.0 (1 ug) 22                                                             ______________________________________                                    

As can be seen by the data, assay amplification was achieved when theanalyte was present at 1 ng or above.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES:  13                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #1:                          - - ATGCGTAGCA GCTTTACCGC AGAGATCATG CCTATGTACC ATGCTATCCT AC -            #CTGTAAGT     60                                                                 - - CATAGCTGTT TCCTG              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #2:                          - - CAGGAAACAG CTATGACTTA CAGGTAGGAT AGCATGGTAC ATAGGCATGA TC -             #TCTGCGGT     60                                                                 - - AAAGCTGCTA CGCAT              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #3:                          - - ATGCGTAGCA GCTTTACCGC AGAGATCATG CCTATGTACC ATGCTATCCT AC -             #CTGTAAGT     60                                                                 - - AAAGCTGCTA CGCAT              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #4:                          - - ATGCGTAGCA GCTTTACTTA CAGGTAGGAT AGCATGGTAC ATAGGCATGA TC -             #TCTGCGGT     60                                                                 - - AAAGCTGCTA CGCAT              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  17 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #5:                          - - ATGCGTAGCA GCTTTAC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  17 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #6:                          - - CAGGAAACAG CTATGAC             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #7:                          - - ATGCGTAGCA GCTTTACCGC AGAGATCATG CCTATGTACC ATGCTATCCT AC -             #CTGTAATA     60                                                                 - - GTAGAAACAG CTGAC              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #8:                          - - GTCAGCTGTT TCTACTATTA CAGGTAGGAT AGCATGGTAC ATAGGCATGA TC -             #TCTGCGGT     60                                                                 - - AAAGCTGCTA CGCAT              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #9:                          - - GGCAGGAAGA CAAACACTGG CTGGTCTGTG GTGCTGTGCT TGTTCCCCTG TC -             #CTAGTATT     60                                                                 - - GTTTTCTGGG TTGGT              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  17 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #10:                         - - ACCAACCCAG AAAACAA             - #                  - #                      - #   17                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  75 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #11:                         - - ACCAACCCAG AAAACAATAC TAGGACAGGG GAACAAGCAC AGCACCACAG AC -             #CAGCCAGT     60                                                                 - - GTTTGTCTTC CTGCC              - #                  - #                      - #    75                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  16 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #12:                         - - GGCAGGAAGA CAAACA             - #                  - #                      - #    16                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH:  17 base - # pairs                                                (B) TYPE:  nucleic a - #cid                                                   (C) STRANDEDNESS:  sing - #le                                                 (D) TOPOLOGY:  linear                                                - -     (ii) MOLECULE TYPE:  DNA (genomic)                                    - -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - #13:                         - - GTCAGCTGTT TCTACTA             - #                  - #                      - #   17                                                                 __________________________________________________________________________

What is claimed is:
 1. A method for simultaneous multiple measurementsof a multiplicity of different immobilized non-nucleic acid analytes ina single test sample comprising the steps of:i) providing multipleimmobilized capture reagents, each comprising at least one member of abinding pair which binds to a nonnucleic acid analyte wherein eachcapture reagent binds, through said at least one member of the bindingpair, to a different non-nucleic acid analyte; ii) providing saidnonnucleic acid analytes and contacting them with said capture agents toform immobilized non-nucleic acid analytes; iii) providing at least onereporter conjugate in the test sample, each conjugate comprising amember of a binding pair conjugated to a unique target nucleic acidsequence wherein each reporter conjugate binds through the member of itsbinding pair to a unique non-nucleic acid analyte thereby forminganalyte-dependent reporter complexes; iv) contacting theanalyte-dependent reporter complexes with a nucleic acid replicationcomposition capable of replicating said target nucleic acid sequences;v) replicating the target nucleic acid sequences; and vi) simultaneouslydetecting the replicated target nucleic acid sequences so as to obtainsimultaneous multiple measurements of said unique nonnucleic acidanalytes.
 2. The method of claim 1, wherein each reporter conjugate iscomprised of a binding pair chemically attached to a target nucleic acidsequence.
 3. The method of claim 2 wherein each target nucleic acidsequence may be differentiated on the basis of sequence length orsequence identity.
 4. The method of claim 3 wherein said targetsequences can be replicated by sets of primers that are specific foreach target sequence.
 5. A method of detecting non-nucleic acid analytescomprising the steps of:a) forming an analyte-dependent reporter complexby contacting an immobilized capture reagent which binds to a nonnucleicacid analyte, with a nonnucleic acid analyte, and a reporter conjugatewherein said reporter conjugate is comprised of a member of a bindingpair which also binds to the nonnucleic acid analyte and is conjugatedto a reporter enzyme; b) contacting the analyte-dependent reportercomplex with a nucleic acid replication substrate comprising a targetnucleic acid sequence conjugated to a moiety capable of detectableactivation by said reporter enzyme to produce a detectably activatednucleic acid replication intermediate which deposits onto an immobilizedreceptor capable of binding to said complex thereby producing adeposited nucleic acid replication product; c) contacting the depositednucleic acid replication product with a nucleic acid replicationcomposition capable of replicating said target nucleic acid sequence; d)replicating a target nucleic acid sequence from the deposited nucleicacid sequences e) detecting said replicated target nucleic acidsequence.
 6. A method of detecting non-nucleic acid analytes comprisingthe steps of:a) forming an analyte-dependent reporter complex bycontacting an immobilized capture reagent which binds to a nonnucleicacid analyte, with a nonnucleic acid analyte, and a reporter conjugatewherein said reporter conjugate is comprised of a member of a bindingpair which also binds to the nonnucleic acid analyte and is conjugatedto a reporter enzyme; b) contacting the analyte-dependent reportercomplex with a nucleic acid replication substrate comprising a targetnucleic acid sequence conjugated to a moiety capable of detectableactivation by said reporter enzyme to produce a detectably activatednucleic acid replication intermediate which deposits onto an immobilizedreceptor capable of binding to said complex thereby producing adeposited nucleic acid replication product; c) contacting the depositednucleic acid replication product with a nucleic acid replicationcomposition capable of replicating said target nucleic acid sequence toproduce a deposited nucleic acid replication binding pair complex, d)replicating a target nucleic acid sequence of the deposited nucleic acidsequences; e) detecting said replicated target nucleic acid sequence. 7.The method of claim 1, further comprising additionally adding to thesample one or more reference nucleic acid sequences and the means forreplicating said reference sequences, whereby said reference sequencesare replicated in addition to said target sequences and thereby servingas internal controls for more accurate detection and quantitation ofmore than one non-nucleic acid analyte and wherein the reference nucleicacid sequences are also attached to binding pairs to form reporterconjugates.
 8. The method of claim 1, wherein the nucleic acidreplication composition provided at step (iv) additionally comprises atleast one replication control comprising reference sequences,replicating said reference sequences at step iv) concurrently with atleast one of said target sequences and at step v) detecting andseparately quantitating the replicated reference sequences andreplicated target sequences, to determine a ratio of the concentrationof replicated reference sequences to the concentration of replicatedtarget sequences, thereby determining from said ratio the concentrationof one or more nonnucleic acid analytes.
 9. The method of claim 1further comprising at step (iii) providing at least one immobilizedreference nucleic acid sequence and at step (iv) contacting the reportercomplexes with said nucleic acid replication composition wherein saidcomposition additionally comprises the means to replicate the referencesequence of said immobilized reference nucleic acid sequences, at step(v) replicating said reference sequences concurrently with said targetsequences and at step (vi) detecting and separately quantitating saidreplicated reference sequences to determine a ratio of the concentrationof the replicated reference sequences to the concentration of replicatedtarget sequences thereby determining from said ratio the concentrationof one or more nonnucleic acid analytes.
 10. The method of claim 1, 2, 4or 7, wherein the nucleic acid sequence replication is accomplishedusing a thermal-stable nucleic acid polymerase.
 11. The method of claim1, 2, 4 or 7, wherein the target or reference nucleic acid sequencecontains at one end a first primer binding sequence, and contains at theother end a sequence which is complementary to the first primer bindingsequence, thereby enabling replication with a single primer.
 12. Themethod of claim 1, 2, 4 or 7, wherein nucleic acid sequence replicationis accomplished using primers which contain sequences at their 5' endswhich are not complementary to the target or reference sequences. 13.The method of claim 1, 2, 4 or 7, wherein nucleic acid sequencereplication is accomplished using a thermal-stable ligase.
 14. Themethod of claim 1, 2, 4 or 7, wherein at least one signal generatingmoiety is incorporated within the replicated nucleic acid sequences. 15.The method of claim 14, wherein said signal-generating moieties areradioactive.
 16. The method of claim 14, wherein said signal-generatingmoieties are luminescent.
 17. The method of claim 14, wherein saidsignal-generating moieties are chemiluminescent.
 18. The method of claim14, wherein said signal generating moieties are enzymes.
 19. The methodof claim 14, wherein said signal generating moieties are fluorescent.20. The method of claim 19, wherein said fluorescent moieties arepositioned within the replicated sequences to enable energy transferbetween said fluorescent moieties.
 21. The method of claim 19, whereinsaid fluorescent moieties are positioned no more than about 12 basesapart within the replicated nucleic acid sequences.
 22. The method ofclaim 1, 2, 4 or 7 wherein primers or ligand labeled bases are labeledwith a first member of a binding pair are incorporated within theamplified nucleic acid sequences.
 23. The method of claim 22, whereindetection and quantitation of the replicated nucleic acid sequences isaccomplished by immobilizing the replicated nucleic acid sequences bydeposition of said sequences onto an immobilized capture reagentcomprising a second member of the binding pair, and further detectingsaid immobilized replicated nucleic sequences.
 24. The method of claim22, wherein said first member of a binding pair is biotin.
 25. Themethod of claim 1, further comprising after step (iii) separating thereplicated nucleic acid sequences from the non-incorporated signalgenerating moieties using size separation techniques; and (iv) detectingthe replicated nucleic acid sequences.
 26. A method for the detectionand quantitation of a nonnucleic acid analyte or analytes comprising:a)adding to an analyte sample an immobilized capture reagent comprising aleast one member of a binding pair; b) adding simultaneously orsequentially with step (a) at least one ligand reporter conjugatecomprising a target nucleic acid sequence conjugated to a ligand whereinthe ligand of the reporter conjugate will compete with the analyte forbinding to the immobilized capture reagent to form immobilized analytecomplexes and/or immobilized ligand complexes; c) washing unboundanalyte and unbound ligand reporter conjugate away from any immobilizedanalyte complex and/or immobilized ligand complexes; d) contactingwashed and immobilized analyte complexes and/or immobilized ligandcomplexes with a nucleic acid replication composition capable ofreplicating the target nucleic acid in any of the immobilized ligandcomplexes; e) detecting the presence of replicated target nucleic acidsequences as a means of determining competitive binding of the ligandreporter conjugate and the analyte to the immobilized capture reagentthereby detecting and quantitating the presence of the nonnucleic acidanalyte or analytes in the sample.
 27. A method for the amplifieddetection of at least one nonnucleic acid analyte comprising the stepsof:a) contacting a nonnucleic acid analyte sample with immobilizedcapture reagent capable of binding to the analyte whereby at least onenonnucleic acid analyte is immobilized; b) adding simultaneously orsequentially with step (a) at least one reporter conjugate to the samplewherein said reporter conjugate comprises a target nucleic acid sequenceconjugated to a member of a binding pair which is capable of binding, tosaid nonnucleic acid analyte; c) separating any excess reporterconjugates which has remained free in solution from the sample; d)contacting free reporter conjugates of step (c) with a nucleic acidreplication composition capable of replicating said target nucleic acid;e) replicating the target nucleic acid sequence of the free reporterconjugates of step (d); f) detecting and quantitating the replicatedtarget nucleic acids of step (e) whereby the presence of at least oneanalyte in the sample is determined.